Humanized IL-4 and IL-4Ra animals

ABSTRACT

Non-human animals comprising a human or humanized IL-4 and/or IL-4Rα nucleic acid sequence are provided. Non-human animals that comprise a replacement of the endogenous IL-4 gene and/or IL-4Rα gene with a human IL-4 gene and/or IL-4Rα gene in whole or in part, and methods for making and using the non-human animals, are described. Non-human animals comprising a human or humanized IL-4 gene under control of non-human IL-4 regulatory elements is also provided, including non-human animals that have a replacement of non-human IL-4-encoding sequence with human IL-4-encoding sequence at an endogenous non-human IL-4 locus. Non-human animals comprising a human or humanized IL-4Rα gene under control of non-human IL-4Rα regulatory elements is also provided, including non-human animals that have a replacement of non-human IL-4Rα-encoding sequence with human or humanized IL-4Rα-encoding sequence at an endogenous non-human C IL-4Rα locus. Non-human animals comprising human or humanized IL-4 gene and/or IL-4Rα sequences, wherein the non-human animals are rodents, e.g., mice or rats, are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/706,319, filed May 7, 2015, which claims the benefit of priority toU.S. Provisional Application No. 61/989,757 filed May 7, 2014, theentire contents of which are incorporated herein by reference.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The Sequence Listing in an ASCII text file, named 31260_SEQ3.txt of 16KB, created on Jul. 24, 2015, and submitted to the United States Patentand Trademark Office via EFS-Web, is incorporated herein by reference.

FIELD OF INVENTION

Non-human animals are disclosed herein which comprise nucleic acidsequences encoding an IL-4 and/or an IL-4Rα protein that comprise ahuman sequence. Transgenic non-human animals are also disclosed hereinwhich comprise an IL-4 and/or an IL-4Rα gene that is human in whole orin part. Non-human animals that express human or humanized IL-4 and/orIL-4Rα proteins are also disclosed. In addition, methods are disclosedfor making and using non-human animals comprising human or humanizedIL-4 and/or IL-4Rα nucleic acid sequences.

BACKGROUND

IL-4 and IL-4Rα are therapeutic targets for treatment of a variety ofhuman diseases, disorders and conditions that are associated withabnormal type-2 T helper (Th2) cells. The evaluation of pharmacokinetics(PK) and pharmacodynamics (PD) of therapeutic molecules thatspecifically target human IL-4 or human IL-4Rα proteins are routinelyperformed in non-human animals, e.g., rodents, e.g., mice or rats.However, the PD of such therapeutic molecules cannot properly bedetermined in certain non-human animals because these therapeuticmolecules do not target the endogenous IL-4 or IL-4Rα proteins.

Moreover, the evaluation of therapeutic efficacy of human-specific IL-4and IL-4Rα protein antagonists using various non-human animal models ofdiseases associated with abnormal Th2 cells is problematic in non-humananimals in which such species-specific antagonists do not interact withthe endogenous IL-4 or IL-4Rα proteins.

Accordingly, there is a need for non-human animals, e.g., rodents, e.g.,murine animals, e.g., mice or rats, in which the IL-4 and/or IL-4Rαgenes of the non-human animal are humanized in whole or in part orreplaced (e.g., at the endogenous non-human loci) with human IL-4 and/orIL-4Rα genes comprising sequences encoding human or humanized IL-4and/or IL-4Rα proteins, respectively.

There is also a need for non-human animals comprising IL-4 and/or IL-4Rαgenes (e.g., humanized, or human) in which the IL-4 and/or IL-4R genesare under control of non-human regulatory elements (e.g., endogenousregulatory elements).

There is also a need for humanized non-human animals that express humanor humanized IL-4 protein in blood, plasma or serum at a concentrationsimilar to that of IL-4 protein present in blood, plasma or serum of anage-matched non-human animal that expresses functional IL-4 protein, butdoes not comprise the human or humanized IL-4 genes, and/or expresshuman or humanized IL-4Rα protein on immune cells, e.g., B and T cells,at a level similar to that of IL-4Rα protein on immune cells, e.g., Band T cells, of an age-matched non-human animal that expressesfunctional IL-4Rα protein, but does not comprise the human or humanizedIL-4Rα gene.

SUMMARY

Non-human animals comprising nucleic acid sequences encoding an IL-4and/or an IL-4Rα protein that comprise a human sequence are provided.

Transgenic non-human animals comprising an IL-4 and/or an IL-4Rα genethat is human in whole or in part are provided.

Non-human animals that express human or humanized IL-4 and/or IL-4Rαproteins are provided.

Non-human animals having a replacement (in whole or in part) ofendogenous non-human animal IL-4 and/or IL-4Rα genes are provided.

Non-human animals comprising an IL-4 and/or an IL-4Rα humanization (inwhole or in part) at an endogenous non-human IL-4 and/or IL-4Rα loci areprovided.

Non-human animals are provided that have a human or humanized IL-4 gene,wherein the non-human animals do not express endogenous IL-4 protein,and wherein the non-human animals express human or humanized IL-4protein in blood, plasma or serum at a concentration similar to that ofIL-4 protein present in blood, plasma or serum of an age-matchednon-human animal that expresses functional endogenous IL-4 protein, butdoes not comprise the human or humanized IL-4 gene.

Non-human animals are provided that have a human or humanized IL-4Rαgene, wherein the non-human animals do not express endogenous IL-4Rαprotein, and express human or humanized IL-4Rα protein on immune cells,e.g., B and T cells, at a level similar to that of IL-4Rα proteinpresent on immune cells, e.g., B and T cells, of an age-matchednon-human animal that expresses functional endogenous IL-4Rα protein,but does not comprise the human or humanized IL-4Rα gene.

In one aspect, non-human animals comprising a human or humanized IL-4and/or IL-4Rα nucleic acid sequence are provided.

In one aspect, genetically modified non-human animals are provided thatcomprise a replacement at an endogenous IL-4 and/or IL-4Rα locus of agene encoding an endogenous IL-4 and/or IL-4Rα with a gene encoding ahuman or humanized IL-4 and/or IL-4Rα protein. Rodents, e.g., mice orrats, are provided that comprise a replacement of an endogenous IL-4gene, at an endogenous rodent IL-4 locus, with a human IL-4 gene, and/orcomprise a replacement of an endogenous IL-4Rα gene, at an endogenousrodent IL-4Rα locus, with a human IL-4Rα gene. In one embodiment, therodent is a mouse. In one embodiment, the rodent is a rat.

In one aspect, genetically modified rodents, e.g., mice or rats, areprovided comprising a humanization of an endogenous rodent IL-4 gene,wherein the humanization comprises a replacement at an endogenous rodentIL-4 locus of a rodent nucleic acid comprising at least one exon of arodent IL-4 gene with a nucleic acid sequence comprising at least oneexon of a human IL-4 gene to form a modified IL-4 gene, whereinexpression of the modified IL-4 gene is under control of rodentregulatory elements at the endogenous rodent IL-4 locus.

In one embodiment, the rodent is a mouse or a rat. In one embodiment,the rodent is a mouse. In one embodiment, the rodent is a rat.

In one embodiment, the modified IL-4 gene encodes human or humanizedIL-4 protein and comprises exon 1 starting from the ATG initiation codonthrough exon 4 of the human IL-4 gene.

In one embodiment, the rodent is a mouse that is incapable of expressinga mouse IL-4 protein.

In one embodiment, the rodent is a mouse that expresses a mouse IL-4Rαprotein encoded by an endogenous mouse IL-4Rα gene.

In one embodiment, the rodent is mouse that expresses a human orhumanized IL-4Rα protein.

In one embodiment, the humanized IL-4Rα protein comprises the ectodomainof a human IL-4Rα protein.

In one embodiment, the humanized IL-4Rα protein comprises thetransmembrane domain and cytoplasmic domain of a mouse IL-4Rα protein.

In one embodiment, the rodent is a mouse that comprises a replacement atan endogenous mouse IL-4Rα locus of a mouse nucleic acid comprising atleast one exon of a mouse IL-4Rα gene with a nucleic acid sequencecomprising at least one exon of a human IL-4Rα gene to form a modifiedIL-4Rα gene, wherein expression of the modified IL-4Rα gene is undercontrol of mouse regulatory elements at the endogenous mouse IL-4Rαlocus.

In one embodiment, the rodent is a mouse, wherein a contiguous genomicfragment of mouse IL-4 sequence comprising exon 1 starting from the ATGinitiation codon through exon 4 of mouse IL-4 is replaced with acontiguous genomic fragment of human IL-4 sequence comprising exon 1starting from the ATG initiation codon through exon 4 of human IL-4.

In one embodiment, expression of the modified IL-4Rα gene encoding thehuman or humanized IL-4Rα protein is under control of mouse regulatoryelements at the endogenous mouse IL-4Rα locus.

In one aspect, genetically modified rodents, e.g., mice or rats, areprovided comprising a humanization of an endogenous rodent IL-4Rα gene,wherein the humanization comprises a replacement at an endogenous rodentIL-4Rα locus of a rodent nucleic acid comprising an exon of a rodentIL-4Rα gene with a nucleic acid sequence encoding at least one exon of ahuman IL-4Rα gene to form a modified (i.e., humanized) IL-4Rα gene,wherein expression of the modified, humanized IL-4Rα gene is undercontrol of rodent regulatory elements at the endogenous rodent IL-4Rαlocus.

In one embodiment, the rodent is a mouse or a rat. In one embodiment,the rodent is a mouse. In one embodiment, the rodent is a rat.

In one embodiment, the modified IL-4Rα gene encodes human or humanizedIL-4Rα protein and comprises exon 1 starting from the ATG initiationcodon through exon 5 of the human IL-4Rα gene.

In one embodiment, the rodent is a mouse that is incapable of expressinga mouse IL-4Rα protein.

In one embodiment, the rodent is a mouse that expresses a mouse IL-4protein encoded by an endogenous mouse IL-4 gene.

In one embodiment, the rodent is mouse that expresses a human orhumanized IL-4 protein.

In one embodiment, the rodent is a mouse that comprises a replacement atan endogenous mouse IL-4 locus of a mouse nucleic acid comprising anexon of a mouse IL-4 gene with a nucleic acid sequence encoding at leastone exon of a human IL-4 gene to form a modified IL-4 gene, whereinexpression of the modified IL-4 gene is under control of mouseregulatory elements at the endogenous mouse IL-4 locus.

In one embodiment, the rodent is a mouse, and wherein a contiguousgenomic fragment of mouse IL-4Rα sequence comprising exon 1 startingfrom the ATG initiation codon through exon 5 of IL-4Rα is replaced witha contiguous genomic fragment of human IL-4Rα sequence comprising exon 1starting from the ATG initiation codon through exon 5 of human IL-4Rα.

In one aspect, genetically modified rodents, e.g., a mouse or rat, areprovided that express a human or humanized IL-4 protein, wherein therodent that expresses a human or humanized IL-4 protein comprises anormal immune system, i.e., the number of immune cells, e.g., B and Tcells, in the blood, plasma or serum of the rodent expressing human orhumanized IL-4 protein are similar to the number of immune cells, e.g.,B and T cells, in the blood, plasma or serum of a rodent that expressesfunctional endogenous IL-4 protein. In one embodiment, the rodent is amouse. In one embodiment, the rodent is a rat.

In one aspect, genetically modified rodents, e.g., a mouse or rat, areprovided that express IL-4 protein from a human or humanized IL-4 gene,wherein the rodent expresses human or humanized IL-4 protein in itsserum. In one embodiment, the rodent is a mouse. In one embodiment, therodent is a rat.

In one embodiment, the serum of the rodent that expresses a human orhumanized IL-4 protein has approximately the same level of IL-4 proteinas a rodent that expresses a functional, endogenous IL-4 protein, e.g.,a wild-type mouse or rat. In one embodiment, the rodent is a mouse. Inone embodiment, the rodent is a rat.

In one embodiment, the mouse expresses human or humanized IL-4 proteinin serum at a concentration of at least about 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%,180%, 190% or 200% of the level of IL-4 protein present in the serum ofan age-matched mouse that expresses functional endogenous IL-4 protein,but does not comprise a replacement of an endogenous IL-4 gene, at anendogenous mouse IL-4 locus, with a human IL-4 gene.

In one embodiment, the mouse expresses human or humanized IL-4 proteinin serum at a concentration of between about 10% and about 200%, betweenabout 20% and about 150%, or between about 30% and about 100% of thelevel of mouse IL-4 protein present in the serum of an age-matched mousethat expresses functional endogenous IL-4 protein, but does not comprisea replacement of an endogenous IL-4 gene, at an endogenous mouse IL-4locus, with a human IL-4 gene.

In one aspect, genetically modified rodents, e.g., a mouse or rat, areprovided that express a human or humanized IL-4Rα protein, wherein therodent expresses a human or humanized IL-4Rα protein on immune cells,e.g., B and T cells, at a level similar to that of IL-4Rα proteinpresent on immune cells, e.g., B and T cells, of an age-matched rodentthat expresses functional endogenous IL-4Rα protein. In one embodiment,the rodent is a mouse. In one embodiment, the rodent is a rat.

In one aspect, genetically modified rodents, e.g., a mouse or rat, areprovided that express IL-4Rα protein from a human IL-4Rα gene, whereinthe rodent expresses human or humanized IL-4Rα protein on immune cells,e.g., B and T cells. In one embodiment, the rodent is a mouse. In oneembodiment, the rodent is a rat.

In one embodiment, the immune cells, e.g., B and T cells, of the rodentthat expresses a human or humanized IL-4Rα protein have approximatelythe same level of IL-4Rα protein on immune cells, e.g., B and T cells,of a rodent that expresses a functional, endogenous IL-4Rα protein,e.g., a wild-type mouse or rat. In one embodiment, the rodent is amouse. In one embodiment, the rodent is a rat.

In one embodiment, the mouse expresses human or humanized IL-4Rα proteinon immune cells, e.g., B and T cells, at an amount of at least about10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%,140%, 150%, 160%, 170%, 180%, 190% or 200% of the amount of IL-4Rαprotein on immune cells, e.g., B and T cells, of an age-matched mousethat expresses functional endogenous IL-4Rα protein, but does notcomprise a replacement of an endogenous IL-4Rα gene, at an endogenousmouse IL-4Rα locus, with a human IL-4Rα gene.

In one embodiment, the mouse expresses human or humanized IL-4Rα proteinon immune cells, e.g., B and T cells, at an amount of between about 10%and about 200%, between about 20% and about 150%, or between about 30%and about 100% of the amount of mouse IL-4Rα protein present on immunecells, e.g., B and T cells, of an age-matched mouse that expressesfunctional endogenous IL-4Rα protein, but does not comprise areplacement of an endogenous IL-4Rα gene, at an endogenous mouse IL-4Rαlocus, with a human IL-4Rα gene.

In one aspect, a genetically modified rodent is provided, comprising ahumanized IL-4Rα gene comprising a replacement of rodent IL-4Rαectodomain-encoding sequence with human IL-4Rα ectodomain-codingsequence, wherein the humanized IL-4Rα gene comprises a rodent IL-4Rαtransmembrane sequence and a rodent IL-4Rα cytoplasmic sequence, whereinthe humanized IL-4Rα gene is under control of endogenous rodent IL-4Rαregulatory elements at the endogenous IL-4Rα locus, and wherein therodent further comprises a humanized IL-4 gene encoding a human orhumanized IL-4 protein, wherein the humanized IL-4 gene is under controlof endogenous rodent IL-4 regulatory elements at the endogenous IL-4locus.

In one embodiment, the rodent is a mouse or a rat. In one embodiment,the rodent is a mouse. In one embodiment, the rodent is a rat.

In one embodiment, the mouse is incapable of expressing a mouse IL-4protein and incapable of expressing a mouse IL-4Rα protein.

In one embodiment, the rodent regulatory elements or sequences at theendogenous rodent IL-4 locus and/or rodent IL-4Rα locus are from a mouseor a rat.

In one embodiment, the rodent regulatory elements or sequences areendogenous rodent regulatory elements or sequences at the rodent IL-4locus and/or rodent IL-4Rα locus.

In one aspect, a non-human animal, e.g., a rodent, e.g., a mouse or rat,is provided that expresses human or humanized IL-4 and/or IL-4Rαproteins, wherein the non-human animal expresses human or humanized IL-4and/or IL-4Rα proteins from an endogenous non-human IL-4Rα locus and/oran endogenous non-human IL-4Rα locus. In an embodiment, the non-humananimal is a rodent. In an embodiment, the rodent is a mouse. In anembodiment, the rodent is a rat.

In one aspect, a genetically modified mouse is provided that expresseshuman or humanized IL-4 protein from an endogenous mouse IL-4 locus,wherein the endogenous mouse IL-4 gene has been replaced, in whole or inpart, with a human IL-4 gene.

In one embodiment, a contiguous mouse genomic nucleic acid of about 6.3kb at an endogenous mouse IL-4 locus, including exon 1 starting from theATG initiation codon through exon 4 (including the 3′ untranslatedregion) and a portion of the 3′ region downstream of exon 4, is deletedand replaced with about 8.8 kb of a human IL-4 nucleic acid sequencecomprising exon 1 starting from the ATG initiation codon through exon 4(including the 3′ untranslated region) and a portion of the 3′ regiondownstream of exon 4 of the human IL-4 gene. In a specific embodiment,the human IL-4 nucleic acid sequence replacing the mouse genomic nucleicacid comprises exon 1 starting from the ATG initiation codon throughexon 4 and a portion of the 3′ region downstream of exon 4 of the humanIL-4 gene of human BAC RP11-17K19. In a specific embodiment, themodified IL-4 gene comprises mouse IL-4 5′ regulatory elements and humanIL-4 exon 1 starting from the ATG initiation codon through exon 4, i.e.,the IL-4 protein coding sequences.

In one aspect, a genetically modified mouse is provided that comprises anucleotide sequence encoding a human or humanized IL-4 protein, whereinthe nucleotide sequence encoding the human or humanized IL-4 proteinreplaces, in whole or in part, an endogenous nucleotide sequenceencoding an endogenous mouse IL-4 protein.

In one aspect, a genetically modified mouse is provided that expresseshuman or humanized IL-4Rα protein from an endogenous mouse IL-4Rα locus,wherein the endogenous mouse IL-4Rα gene has been replaced, in whole orin part, with a human IL-4Rα gene.

In one embodiment, a contiguous mouse genomic nucleic acid of about 7.1kb at the endogenous mouse IL-4Rα locus, including exon 1 starting fromthe ATG initiation codon through exon 5 and a portion of intron 5, isdeleted and replaced with about 15.6 kb of human IL-4Rα nucleic acidsequence comprising exon 1 starting from the ATG initiation codonthrough exon 5 and a portion of intron 5 of the human IL-4Rα gene. In aspecific embodiment, the human IL-4α nucleic acid replacing the mousegenomic nucleic acid comprises exon 1 starting from the ATG initiationcodon through exon 5 and a portion of intron 5 of the human IL-4α geneof human BAC RP11-16E24. In a specific embodiment, the human IL-4Rαnucleic acid replacing the mouse genomic nucleic acid comprises theentire human IL-4Rα ectodomain coding sequence.

In one aspect, a method is provided for making a humanized IL-4 rodent,comprising replacing a rodent IL-4 gene sequence encoding rodent IL-4protein with a human IL-4 nucleic acid sequence comprising one or moreexons of the human IL-4 gene sequence to form a modified, humanized IL-4gene encoding human or humanized IL-4 protein, wherein the replacementis at an endogenous rodent IL-4 locus and the humanized IL-4 genesequence comprising one or more exons of the human IL-4 gene sequenceand encoding human or humanized IL-4 protein is operably linked torodent regulatory elements or sequences (e.g., 5′ and/or 3′ regulatoryelements) at the endogenous rodent IL-4 locus.

In one embodiment, the rodent is a mouse or a rat. In one embodiment,the rodent is a mouse. In one embodiment, the rodent is a rat.

In one embodiment, the rodent regulatory elements or sequences arederived from a mouse. In one embodiment, the rodent regulatory elementsor sequences are derived from a rat.

In one embodiment, the rodent regulatory elements or sequences areendogenous rodent regulatory elements or sequences at the rodent IL-4locus. In one embodiment, the rodent is a mouse. In one embodiment, therodent is a rat.

In one embodiment, the human IL-4 nucleic acid sequence replacing therodent IL-4 gene sequence comprises at least one exon of the human IL-4gene sequence. In other embodiments, the human IL-4 nucleic acidsequence replacing the rodent IL-4 gene sequence comprises at least 2 orat least 3 exons of the human IL-4 gene sequence. In one embodiment, thehuman IL-4 nucleic acid sequence replacing the rodent IL-4 gene sequencecomprises all 4 exons of the human IL-4 gene sequence. In oneembodiment, the rodent is a mouse. In one embodiment, the rodent is arat.

In one embodiment, the human IL-4 nucleic acid sequence replacing therodent IL-4 gene sequence encodes a protein that is about 85%, 90%, 95%,96%, 97%, 98%, or about 99% identical to a human IL-4 (e.g., the humanIL-4 protein encoded by the nucleic acid set forth in GenBank AccessionNo. NM_000589.3s).

In one embodiment, the replacement is at an endogenous rodent IL-4 locusand the humanized IL-4 gene sequence comprising one or more exons of thehuman IL-4 gene sequence and encoding human or humanized IL-4 protein isoperably linked to endogenous rodent regulatory elements or sequences(e.g., 5′ and/or 3′ regulatory elements) at the endogenous rodent IL-4locus.

In one aspect, a method is provided for making a humanized IL-4 mouse,comprising replacing a mouse IL-4 gene sequence encoding mouse IL-4protein with a human IL-4 gene sequence to form a modified, humanizedIL-4 gene encoding human or humanized IL-4 protein.

In one embodiment, the replacement is at an endogenous mouse IL-4 locus,and the resulting humanized IL-4 gene encoding human or humanized IL-4protein is operably linked to mouse regulatory elements or sequences(e.g., 5′ and/or 3′ regulatory elements) at the endogenous mouse IL-4locus.

In one embodiment, the replacement is at an endogenous mouse IL-4 locus,and the humanized IL-4 gene encoding human or humanized IL-4 protein isoperably linked to endogenous mouse regulatory elements or sequences(e.g., 5′ and/or 3′ regulatory elements) at the endogenous mouse IL-4locus.

In one aspect, a method is provided for making a humanized IL-4Rαrodent, comprising replacing a rodent IL-4Rα gene sequence encodingrodent IL-4Rα protein with a human IL-4Rα nucleic acid sequencecomprising one or more exons of the human IL-4Rα gene sequence to form amodified, humanized IL-4Rα gene encoding human or humanized IL-4Rαprotein, wherein the replacement is at an endogenous rodent IL-4Rα locusand the humanized IL-4Rα gene sequence comprising one or more exons ofthe human IL-4Rα gene sequence and encoding human or humanized IL-4Rαprotein is operably linked to rodent regulatory elements or sequences(e.g., 5′ and/or 3′ regulatory elements) at the endogenous rodent IL-4Rαlocus.

In one embodiment, the rodent is a mouse or a rat. In one embodiment,the rodent is a mouse. In one embodiment, the rodent is a rat.

In one embodiment, the rodent regulatory elements or sequences arederived from a mouse. In one embodiment, the rodent regulatory elementsor sequences are derived from a rat.

In one embodiment, the rodent regulatory elements or sequences areendogenous rodent regulatory elements or sequences at the rodent IL-4Rαlocus. In one embodiment, the rodent is a mouse. In one embodiment, therodent is a rat.

In one embodiment, the human IL-4Rα nucleic acid sequence replacing therodent IL-4Rα gene sequence comprises at least one exon of the humanIL-4Rα gene sequence. In other embodiments, the human IL-4Rα nucleicsequence replacing the rodent IL-4Rα gene sequence comprises at least 2,3, 4, 5, 6, 7, or 8 exons of the human IL-4Rα gene sequence. In oneembodiment, the human IL-4Rα nucleic sequence replacing the rodentIL-4Rα gene sequence comprises all 9 exons of the human IL-4Rα genesequence. In one embodiment, the rodent is a mouse. In one embodiment,the rodent is a rat.

In one embodiment, the human IL-4Rα nucleic sequence replacing therodent IL-4Rα gene sequence encodes a protein that is about 85%, 90%,95%, 96%, 97%, 98%, or about 99% identical to a human IL-4Rα (e.g., thehuman IL-4Rα protein encoded by the nucleic acid set forth in GenBankAccession No. NM_000418.3).

In one embodiment, the human IL-4Rα nucleic acid sequence replacing therodent IL-4Rα gene sequence comprises at least one exon of the humanIL-4Rα gene sequence encoding the ectodomain of the human IL-4Rαprotein. In other embodiments, the human IL-4Rα nucleic acid sequencereplacing the rodent IL-4Rα gene sequence comprises at least 2, 3, or 4exons of the human IL-4Rα gene sequence encoding the ectodomain of thehuman IL-4Rα protein. In one embodiment, the human IL-4Rα nucleic acidsequence replacing the rodent IL-4Rα gene sequence comprises all 5 exonsof the human IL-4Rα gene sequence encoding the ectodomain of the humanIL-4Rα protein. In one embodiment, the rodent is a mouse. In oneembodiment, the rodent is a rat.

In one embodiment, the human or humanized IL-4Rα gene sequence replacingthe rodent IL-4Rα gene sequence encodes an ectodomain of the IL-4Rαprotein that is about 85%, 90%, 95%, 96%, 97%, 98%, or about 99%identical to the ectodomain of a human IL-4Rα protein (e.g., the humanIL-4Rα protein encoded by the nucleic acid set forth in GenBankAccession No. NM_000418.3).

In one embodiment, the replacement is at an endogenous rodent IL-4Rαlocus and the humanized IL-4Rα gene sequence comprising one or moreexons of the human IL-4Rα gene sequence and encoding human or humanizedIL-4Rα protein is operably linked endogenous rodent regulatory elementsor sequences (e.g., 5′ and/or 3′ regulatory elements) at the endogenousrodent IL-4Rα locus.

In one aspect, a method is provided for making a humanized IL-4Rα mouse,comprising replacing a mouse IL-4Rα gene sequence encoding mouse IL-4Rαprotein with a human IL-4Rα nucleic acid sequence to form a humanizedIL-4Rα gene encoding human or humanized IL-4Rα protein.

In one embodiment, the replacement is at an endogenous mouse IL-4Rαlocus, and the humanized IL-4Rα gene encoding human or humanized IL-4Rαprotein is operably linked to mouse regulatory elements or sequences(e.g., 5′ and/or 3′ regulatory elements) at the endogenous mouse IL-4Rαlocus.

In one embodiment, the replacement is at an endogenous mouse IL-4Rαlocus, and the humanized IL-4Rα gene encoding human or humanized IL-4Rαprotein is operably linked to endogenous mouse regulatory elements orsequences (e.g., 5′ and/or 3′ regulatory elements) at the endogenousmouse IL-4Rα locus.

In various aspects, the genetically modified non-human animals, e.g.,rodents, e.g., mice or rats, described herein comprise the geneticmodifications in their germ-line.

In one aspect, a non-human animal, e.g., rodent, e.g., a mouse or rat,embryo comprising a genetic modification as described herein isprovided.

In one aspect, a non-human animal, e.g., rodent, e.g. a mouse or rat,host embryo is provided that comprises a donor cell that comprises agenetic modification as described herein.

In one aspect, a pluripotent or totipotent non-human animal, e.g.,rodent, e.g., mouse or rat, cell comprising a genetic modification asdescribed herein is provided. In one embodiment, the cell is a rodentcell. In one embodiment, the cell is a mouse cell. In one embodiment,the cell is a rodent ES cell. In one embodiment, the cell is a mouse EScell.

In one aspect, a non-human animal, e.g., rodent, e.g., mouse or rat, eggis provided, wherein the non-human animal egg comprises an ectopicnon-human animal chromosome, wherein the ectopic non-human animalchromosome comprises a genetic modification as described herein. In oneembodiment, the non-human animal is a rodent. In one embodiment, therodent is a mouse. In one embodiment, the rodent is a rat.

In one aspect, the mouse embryo, egg, or cell that is geneticallymodified to comprise a human IL-4 gene or human IL-4Rα gene is of amouse that is of a C57BL strain selected from C57BL/A, C57BL/An,C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ,C57BL/10, C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola. In anotherembodiment, the mouse is a 129 strain selected from the group consistingof a strain that is 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1/SV,129S1/Svlm), 129S2, 129S4, 129S5, 129S9/SvEvH, 129S6 (129/SvEvTac),129S7, 129S8, 129T1, 129T2 (see, e.g., Festing et al. (1999) Revisednomenclature for strain 129 mice, Mammalian Genome 10:836, see also,Auerbach et al (2000) Establishment and Chimera Analysis of 129/SvEv-and C57BL/6-Derived Mouse Embryonic Stem Cell Lines). In a specificembodiment, the genetically modified mouse is a mix of an aforementioned129 strain and an aforementioned C57BL/6 strain. In another specificembodiment, the mouse is a mix of aforementioned 129 strains, or a mixof aforementioned BL/6 strains. In a specific embodiment, the 129 strainof the mix is a 129S6 (129/SvEvTac) strain. In another embodiment, themouse is a BALB strain, e.g., BALB/c strain. In yet another embodiment,the mouse is a mix of a BALB strain and another aforementioned strain.In one embodiment, the mouse is Swiss or Swiss Webster mouse.

In various aspects, the non-human animals comprising a human orhumanized IL-4R and/or IL-4 nucleic acid sequence are selected frommammals and birds. In one embodiment, the non-human animals are mammals.In one embodiment, the mammals are murine.

In one aspect, a method of screening for a human-specific IL-4 or IL-4Rαantagonist is provided. The method is useful for identifying therapeuticcandidates and evaluating therapeutic efficacy. The method comprisesadministering an agent to a genetically modified rodent that is doublyhumanized for IL-4 and IL-4Rα as described herein, determining an effectof the agent on a biological function mediated by the IL-4/IL-4Rαsignaling pathway, and identifying the agent as a human-specific IL-4 orIL-4Rα antagonist if it antagonizes the function mediated by theIL-4/IL-4Rα signaling pathway in the genetically modified rodent.

In one embodiment, the agent comprises an immunoglobulin variable domainthat binds IL-4 or IL-4Rα. In one embodiment, the agent specificallybinds human IL-4 or IL-4Rα, but not rodent IL-4 or IL-4Rα. In oneembodiment, the agent is an antibody. In a specific embodiment, theagent is an antibody that specifically binds human IL-4Rα, but notrodent IL-4Rα.

In one embodiment, the screening method utilizes a doubly humanizedmouse that expresses a human IL-4 protein, and a humanized IL-4Rαprotein, wherein the humanized IL-4Rα protein includes the ectodomain ofa human IL-4Rα protein, linked to the transmembrane and cytoplasmicdomains of the endogenous mouse IL-4Rα protein, and wherein the mousedoes not express murine IL-4 or murine IL-4Rα.

In some embodiments, the method of screening includes the steps ofinducing in a doubly humanized rodent as described herein a diseaseassociated with IL-4/IL-4Rα signaling, administering an agent to therodent, determining whether the agent ameliorates the disease, andidentifying the agent as a human-specific IL-4 or IL-4Rα antagonistsuitable for treating the disease if the agent ameliorates the disease.

In some embodiments, the disease associated with IL-4/IL-4Rα signalingis airway inflammation, which can be induced in a rodent by intranasaladministration of an allergen (e.g., house dust mite extract) in one ormore doses for a period of time. The effect of an agent can bedetermined by measuring whether the extent of airway inflammation(reflected by e.g., mucus accumulation, infiltrating cells inbronchoalveolar lavage fluid, and/or levels of total circulating IgE),is reduced as a result of the administration of the agent.

In some embodiments, the disease associated with IL-4/IL-4Rα signalingis skin inflammation or atopic dermatitis, which can be induced in arodent by creating skin injury and exposing the injured skin to anallergen (e.g., bacterial toxin or house dust mite extract) in one ormore doses for a period of time. The effect of an agent can bedetermined by measuring whether skin inflammation is reduced as a resultof the administration of the agent.

In a further aspect, a triply humanized non-human animal whose IL-4,IL-4Rα, and IL-33 genes have been humanized as described herein is usedto evaluate the pharmacodynamics (PD) and therapeutic efficacy of acompound or a combination of compounds.

Each of the aspects and embodiments described herein are capable ofbeing used together, unless excluded either explicitly or clearly fromthe context of the embodiment or aspect.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides an illustration, not to scale, of the receptors for IL-4and IL-13 signal transduction and the mechanism of action of dupilumab,a neutralizing fully human monoclonal antibody that binds specificallyto the human IL-4 receptor α chain (IL-4Rα).

FIGS. 2A-2B provide an illustration, not to scale, of the strategies forhumanization of the IL-4 (Il4) and IL-4Rα (Il4ra) loci. (2A) The mouseIL-4 gene (top) spanning the coding region from exon 1 starting at theATG initiation codon through exon 4 (including the 3′ untranslatedregion) and a portion of the 3′ region downstream of exon 4 are deletedand replaced by the coding region from exon 1 starting at the ATG codonthrough exon 4 (including the 3′ untranslated region) and a portion ofthe 3′ region downstream of exon 4 of the human IL-4 gene (bottom) alongwith a floxed hygro selection cassette loxP, as indicated. (2B) Themouse IL-4Rα gene (top) spanning the coding region from exon 1 startingfrom the ATG initiation codon through exon 5 and a portion of intron 5are deleted and replaced by the coding region from exon 1 starting fromthe ATG initiation coding through exon 5 and a portion of intron 5 ofthe human IL-4Rα gene (bottom) and a floxed neo selection cassette, asindicated.

FIG. 3 shows the expression of humanized IL-4Rα protein on B and T cellsfrom doubly humanized IL-4/IL-4Rα (Il4^(hu/hu)/Il4ra^(hu/hu)) mice.

FIG. 4 shows the IL-4 and IL-13 ligand specificities and receptorfunctionalities using primary cells derived from humanized IL4Rα(Il4ra^(hu/hu)) mice.

FIG. 5 shows IL-4 dependent IgE production in vivo in wild-type, but nothumanized IL4Rα (Il4ra^(hu/hu)) mice.

FIG. 6 shows that dose-dependent IL-4 induction of mIgE production exvivo in mouse B cells (left panel) is blocked by dupilumab, in adose-dependent manner (right panel).

FIG. 7 shows that dupilumab, in a dose-dependent manner, prevents IL-25induced lung pathologies in vivo in humanized IL4Rα (Il4ra^(hu/hu))mice.

FIG. 8 shows the experimental design for assessing the therapeuticefficacy of dupilumab in a house dust mite extract (HDM) inducedpulmonary inflammation model using doubly humanized IL-4 and IL-4Rα(IL-4^(hu/hu)/IL-4R^(hu/hu)) mice. “REGN668” refers to a humanmonoclonal antibody directed to human IL-4Rα, also known as dupilumab.“REGN129” refers to a mouse sol IL-13Rα2-Fc, which is a fusion proteinbetween the ectodomain of mouse IL-13R2α and Fc.

FIG. 9 shows the experimental design for assessing the therapeuticefficacy of dupilumab in an HDM induced pulmonary inflammation modelusing doubly humanized IL-4 and IL-4Rα (Il-4^(hu/hu)/Il-4R^(hu/hu)) miceand an isotype control antibody.

FIGS. 10A-10C illustrate the strategies for humanization of the mouseIL-33 locus. FIG. 10A illustrates that the mouse IL-33 gene (top)spanning the coding region from exon 2 starting at the ATG initiationcodon through the stop codon in exon 8 is deleted and replaced by thecoding region from exon 2 starting at the ATG codon through exon 8(including the 3′ untranslated region) of the human IL-33 gene (bottom).FIG. 10B shows the humanized IL-33 allele in mouse ES cell clone MAID7060, which contains a loxP neomycin selection cassette. FIG. 10C showsthe humanized IL-33 allele in mouse ES cell clone MAID 7061, in whichthe neomycin selection cassette has been deleted, with loxP and cloningsites (77 bp) remaining downstream of the human IL-33 sequence, andmouse 3′ UTR retained downstream of the loxP site.

DETAILED DESCRIPTION

IL-4 and IL-4Rα as Therapeutic Targets

Allergic disorders are a spectrum of diseases that are occurring at anincreasing rate, especially in developed countries. Atopic dermatitis,asthma, and allergic rhinitis are the most common inflammatoryconditions among patients with allergies; these patients often sufferthe onset of multiple clinical symptoms. The pathogenesis of allergy islinked to abnormal immune responses against exogenous antigens (seeMueller et al. (2002) Structure, binding, and antagonists in theIL-4/IL-13 receptor system, Biochim Biophys Acta 1592:237-250).

Over-production of antigen-specific IgE is an essential component totrigger allergic inflammation. Abnormal type-2 T helper cell (Th2)polarization contributes to the increased IgE responses.

Interleukin-4 (IL-4) and interleukin-13 (IL-13), originally identifiedfrom activated T cells, are major Th2 cytokines that play central rolesin initiating and sustaining the immune and inflammatory reactions inallergies.

IL-4 and IL-13 signaling are mediated by two distinct receptor complexeswith a shared subunit, IL-4 receptor alpha (IL-4Rα), which maycontribute to the overlapping biological responses between these twocytokines. See FIG. 1.

Receptors for Interleukin-4/13 Signal Transduction and the Mechanism ofAction of Dupilumab.

IL-4Rα forms two distinct heterodimeric receptor complexes to mediatethe biological functions of IL-4 and IL-13 in a tissue- andresponse-specific manner. The type I receptor comprised of IL-4Rα andcommon cytokine receptor gamma chain (γC) is unique for IL-4. Type IIreceptor formed between IL-4Rα and IL-13Rα1 is the primary receptor forIL-13, but is also functional for IL-4. In addition, IL-13 will bind toa second high affinity receptor, IL-13Rα2, which is generally recognizedas a decoy receptor or with a possible, pro-fibrotic effect in thefull-length form.

Dupilumab is an antagonistic monoclonal antibody against human IL-4Rαthat inhibits induced biological activities from IL-4 and IL-13.Dupilumab blocks IL-4 signal transduction by preventing its binding toreceptor subunits, whereas the inhibitory effect on IL-13 signaling islikely mediated through interfering with the dimeric receptorinteraction.

Dupilumab, a fully-human monoclonal antibody directed against the sharedIL-4Rα subunit, was developed at Regeneron Pharmaceuticals, Inc. usingVelocImmune® mice. Dupilumab is undergoing clinical trials for thetreatment of moderate-to-severe asthma and for the treatment ofmoderate-to-severe atopic dermatitis.

Evaluating the potency of dupilumab in murine models presents multiplechallenges: (a) dupilumab does not recognize the cognate mouse IL-4receptor; and (b) there is a lack of functional interaction betweenmouse IL-4 protein and human IL-4 receptor.

IL-4 Gene and Protein

The IL-4 gene encodes a secreted IL-4 protein, which plays an importantrole in the activation of B cells, as well as other cell types (see FIG.1).

Human IL-4.

NCBI Gene ID: 3565; Primary source: HGNC:6014; RefSeq transcript:NM_000589.3; UniProt ID: P05112; Genomic assembly: GRCh37; Location:chr5:132,009,743-132,018,576+ strand.

The human IL-4 gene is located on chromosome 5, at 5q31.1. The humanIL-4 gene has 4 exons and encodes a precursor polypeptide of 153 aminoacids in length, including a 24 amino acid signal peptide, and a 129amino acid mature IL-4 protein.

Mouse IL-4.

NCBI Gene ID: 16189; Primary source: MGI:96556; RefSeq transcript:NM_021283.2; UniProt ID: P07750; Genomic assembly: GRCm38; Location:chr11:53,612,350-53,618,606− strand.

The mouse IL-4 gene is located on chromosome 11, at 11 31.97 cM. Themouse IL-4 gene has 4 exons and encodes a precursor polypeptide of 140amino acids in length, including a 20 amino acid signal peptide, and a120 amino acid mature IL-4 protein.

IL-4Rα Gene and Protein

The IL-4Rα gene encodes the transmembrane receptor IL-4Rα protein, whichis expressed primarily on B and T cells, is a receptor for the IL-4 andIL-13 proteins (see FIG. 1).

Human IL-4Rα.

NCBI Gene ID: 3566; Primary source: MGI:6015; RefSeq transcript:NM_000418.3; UniProt ID: P24394; Genomic assembly: GRCh37; Location:chr16:27,351,525-27,367,111+ strand.

The human IL-4Rα gene is located on chromosome 16 at 16p12.1-p11.2. Thehuman IL-4Rα gene has 9 coding exons and encodes a precursor polypeptideof 825 amino acids, including a 25 amino acid signal peptide, and an 800amino acid mature IL-4Rα protein, with the first 207 amino acid residuesof the mature protein constituting the extracellular domain. Theextracellular domain (i.e., ectodomain) of the human IL-4Rα protein isencoded by coding exons 1 through 5 of the human IL-4Rα gene.

Mouse IL-4Rα.

NCBI Gene ID: 16190; Primary source: MGI:105367; RefSeq transcript:NM_001008700.3; UniProt ID: P16382; Genomic assembly: GRCm38; Location:chr11:125,565,655-125,572,745+ strand.

The mouse IL-4Rα gene is located on chromosome 7 at 7 68.94 cM. Themouse IL-4Rα gene has 9 coding exons and encodes a precursor polypeptideof 810 amino acids, including a 25 amino acid signal peptide, and a 785amino acid mature IL-4Rα protein, with the first 208 amino acid residuesof the mature protein constituting the extracellular domain. Theextracellular domain (i.e., ectodomain) of the mouse IL-4Rα protein isencoded by coding exons 1 through 5 of the mouse IL-4Rα gene.

Species Specificity of IL-4 and IL-4Rα Proteins

As shown herein, mouse, but not human, IL-4 is functional in wild-typemice, and, conversely, human, but not mouse, IL-4 is functional inhumanized IL-4Rα (Il4ra^(hu/hu)) mice. (See also, e.g., Andrews et al.(2001) Reconstitution of a functional human type II IL-4/IL-13 receptorin mouse B cells: demonstration of species specificity, J Immunol.166:1716-1722).

Species Specificity of Human IL-4 and IL-4Rα Inhibitors

Candidate therapeutic molecules that target the IL-4 or IL-4Rα proteinsare typically evaluated for pharmacokinetics (PK) and pharmacodynamics(PD) in non-human animals, e.g., rodents, e.g., mice or rats. Suchtherapeutic molecules are also tested for in vivo therapeutic efficacyin non-human animal, e.g., rodent, e.g., mouse or rat, models of humandiseases, disorders and conditions associated with abnormal Th2 cells.

However, therapeutic molecules that are specific for the human IL-4 orIL-4Rα proteins, e.g., human-specific IL-4 or IL-4Rα inhibitors, cannotbe adequately evaluated for PD or in vivo therapeutic efficacy inrodents, in particular mice, because the targets of these therapeuticmolecules are missing. This problem is not overcome using transgenicnon-human animals, e.g., rodents, e.g., mice or rats, expressing humanIL-4 or IL-4Rα proteins because of the above-mentioned speciesspecificity of IL-4 protein.

Accordingly, in various embodiments, to assess the PD and in vivotherapeutic efficacy of a human-specific IL-4 or IL-4Rα proteinantagonist or inhibitor in non-human animals, e.g., rodents, e.g., miceor rats, it is desirable to replace the endogenous IL-4 and/or IL-4Rαproteins with human IL-4 and/or IL-4Rα proteins.

Further, in various embodiments, in order to avoid potential problems ofover- or under-expression of the human IL-4 and/or IL-4Rα proteins, itis desirable to insert the human IL-4 and/or IL-4Rα genes into thegenome of the non-human animals, e.g., rodents, e.g., mice or rats, atthe endogenous IL-4 and/or IL-4Rα gene loci, and to express the humanIL-4 and/or IL-4Rα proteins in non-human animals, e.g., rodents, e.g.,mice or rats, under the control, at least in part, of the endogenousIL-4 and/or IL-4Rα regulatory elements.

Genetically Modified Non-Human Animals

Genetically modified non-human animals are provided herein whoseendogenous IL-4 gene and/or IL-4Rα gene has been replaced in whole or inpart, at an endogenous IL-4 locus and/or the IL-4Rα locus, with a humanIL-4 nucleic acid and/or human IL-4Rα nucleic acid to form a modifiedIL-4 gene and/or modified IL-4Rα gene which encodes a human or humanizedIL-4 and/or human or humanized IL-4Rα protein.

The phrase “non-human animal” as used herein refers to any vertebrateorganism that is not a human. In some embodiments, the non-human animalis a mammal. In specific embodiments, the non-human animal is a rodentsuch as a rat or a mouse.

In one aspect, genetically modified rodents, e.g., mice or rats, areprovided whose endogenous rodent IL-4 gene has been replaced in whole orin part, at an endogenous IL-4 locus, with a human IL-4 nucleic acid.

The replacement involves a replacement of at least one exon, i.e., oneor more exons, of a rodent IL-4 gene with a human nucleic acidcomprising at least one exon of a human IL-4 gene. In some embodiments,a contiguous rodent genomic fragment which includes exon 1 starting fromthe ATG initiation codon through exon 4 of a rodent IL-4 gene has beenreplaced with a contiguous human genomic fragment including exon 1starting from the ATG initiation codon through exon 4 of a human IL-4gene. In a specific embodiment, the rodent is a mouse, and a contiguousmouse genomic fragment of about 6.3 kb at an endogenous mouse IL-4locus, including exon 1 starting from the ATG initiation codon throughexon 4 (including the 3′ untranslated region) and a portion of the 3′region downstream of exon 4, is deleted and replaced with about 8.8 kbof a human IL-4 nucleic acid sequence comprising exon 1 starting fromthe ATG initiation codon through exon 4 (including the 3′ untranslatedregion) and a portion of the 3′ region downstream of exon 4 of the humanIL-4 gene.

In some embodiments, the replacement results in a modified, humanizedIL-4 gene at an endogenous IL-4 gene locus, wherein the expression ofthe modified IL-4 gene is under control of the endogenous regulatoryelements at the endogenous IL-4 locus. The term “regulatory elements” asused herein refer to transcriptional regulatory sequences, includingboth 5′ transcriptional regulatory sequences such as promoter, enhancer,and suppressor elements, and 3′ transcriptional regulatory sequencessuch as a transcriptional termination sequence. In some embodiments, theexpression of a modified IL-4 gene is under control of the endogenous 5′regulatory elements. In other embodiments, the expression of a modifiedIL-4 gene is under control of the endogenous 3′ regulatory elements. Incertain embodiments, the expression of a modified IL-4 gene is undercontrol of the endogenous 5′ and 3′ regulatory elements.

The modified, humanized IL-4 gene formed at an endogenous IL-4 locusencodes a human or humanized IL-4 protein. The term “humanized” refer tonucleic acids or proteins which include portions or sequences of a geneor protein found in a non-human animal (e.g., a rodent such as mouse orrat), and also include portions or sequences that differ from thosefound in a non-human animal but instead correspond to (identical with)portions or sequences of the counterpart human gene or protein. Themodified, humanized IL-4 gene can encode an IL-4 protein that is atleast 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100%identical, with a human IL-4 protein (e.g., the human IL-4 proteinencoded by the nucleic acid set forth in GenBank Accession No.NM_000589.3).

A genetically modified rodent having a replacement of an endogenousrodent IL-4 gene in whole or in part, at an endogenous IL-4 locus, witha human IL-4 nucleic acid, can be homozygous or heterozygous withrespect to the replacement. In some embodiments, the geneticallymodified rodent is heterozygous with respect to the replacement, i.e.,only one of the two copies of the endogenous rodent IL-4 gene has beenreplaced with a human IL-4 nucleic acid. In other embodiments, thegenetically modified rodent is homozygous with respect to thereplacement, i.e., both copies of the endogenous rodent IL-4 gene havebeen replaced with a human IL-4 nucleic acid.

The genetically modified rodent expresses a human or humanized IL-4protein in the serum. In some embodiments, the genetically modifiedrodent does not express endogenous rodent IL-4 protein. In oneembodiment, the serum of the rodent that expresses a human or humanizedIL-4 protein has approximately the same level of IL-4 protein as arodent that expresses a functional, endogenous IL-4 protein, e.g., awild-type rodent (e.g., a rodent that expresses functional endogenousIL-4 protein, but does not comprise a replacement of an endogenous IL-4gene in whole or in part, at an endogenous IL-4 locus, with a human IL-4nucleic acid). By “approximately the same level” it is meant a levelthat falls within 25%, 20%, 15%, 10%, 5% or less in either direction(i.e., greater than or less than) of the level in a wild-type rodent. Inother embodiments, the rodent expresses a human or humanized IL-4protein in serum at a concentration of at least about 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%,170%, 180%, 190% or 200% of the level of IL-4 protein present in theserum of an age-matched rodent that expresses functional endogenous IL-4protein, but does not comprise a replacement of an endogenous IL-4 genein whole or in part, at an endogenous IL-4 locus, with a human IL-4nucleic acid.

In some embodiments, a genetically modified rodent having a replacementof an endogenous rodent IL-4 gene in whole or in part with a human IL-4nucleic acid and expressing a human or humanized IL-4 protein in theserum has a normal immune system, i.e., the number of immune cells,e.g., B and T cells, in the blood, plasma or serum of the rodentexpressing human or humanized IL-4 protein are similar to the number ofimmune cells, e.g., B and T cells, in the blood, plasma or serum of arodent that expresses functional endogenous IL-4 protein and does nothave a replacement of an endogenous rodent IL-4 gene in whole or in partwith a human IL-4 nucleic acid.

In additional embodiments, a genetically modified rodent having areplacement of an endogenous rodent IL-4 gene in whole or in part with ahuman IL-4 nucleic acid and expressing a human or humanized IL-4protein, also includes a replacement of the endogenous rodent IL-4Rαgene in whole or in part, at an endogenous IL-4Rα locus, with a humanIL-4Rα nucleic acid, and as a result, also expresses a human orhumanized IL-4Rα protein.

In another aspect, genetically modified rodents, e.g., mice or rats, areprovided whose endogenous rodent IL-4Rα gene has been replaced in wholeor in part, at an endogenous IL-4Rα locus, with a human IL-4Rα nucleicacid.

The replacement involves replacement of at least one exon, i.e., one ormore exons, of a rodent IL-4Rα gene with a human nucleic acid comprisingat least one exon of a human IL-4Rα gene. In some embodiments, thereplacement involves replacement of at least one of the exons of arodent IL-4Rα gene encoding the rodent ectodomain with at least one ofthe exons of human IL-4Rα gene encoding the human ectodomain. In someembodiments, the replacement involves replacement with a human nucleicacid comprising at least 2, 3 or 4 of the 5 exons encoding theectodomain of a human IL-4Rα gene. In other embodiments, a contiguousrodent genomic fragment which includes exon 1 starting from the ATGinitiation codon through exon 5 of a rodent IL-4Rα gene has beenreplaced with a genomic fragment including exon 1 starting from the ATGinitiation codon through exon 5 of a human IL-4Rα gene. In a specificembodiment, the rodent is a mouse, and a contiguous mouse genomicfragment of about 7.1 kb at an endogenous mouse IL-4Rα locus, includingexon 1 starting from the ATG initiation codon through exon 5 and aportion of intron 5, is deleted and replaced with about 15.6 kb of ahuman IL-4Rα nucleic acid sequence comprising exon 1 starting from theATG initiation codon through exon 5 and a portion of intron 5 of thehuman IL-4Rα gene.

In some embodiments, the replacement results in a modified, humanizedIL-4Rα gene at an endogenous IL-4Rα gene locus, wherein the expressionof the modified IL-4Rα gene is under control of the endogenousregulatory elements at the endogenous IL-4Rα locus.

The modified, humanized IL-4Rα gene formed at an endogenous IL-4Rα locusencodes a human or humanized IL-4Rα protein. In some embodiments, themodified, humanized IL-4Rα gene encodes an IL-4Rα protein that is atleast 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100%identical, with a human IL-4Rα protein (e.g., the human IL-4 Ra proteinencoded by the nucleic acid set forth in GenBank Accession No.NM_000418.3). In other embodiments, the modified IL-4Rα gene encodes ahumanized IL-4Rα protein which comprises an ectodomain that is at least85%, 90%, 95%, 96%, 97%, 98%, or 99% identical, or is 100% identical,with the ectodomain of a human IL-4Rα protein (e.g., the human IL-4Rαprotein encoded by the nucleic acid set forth in GenBank Accession No.NM_000418.3). In specific embodiments, the transmembrane and cytoplasmicdomains of the humanized IL-4Rα protein are identical with thetransmembrane and cytoplasmic domains of the endogenous rodent IL-4Rαprotein.

A genetically modified rodent having a replacement of an endogenousrodent IL-4Rα gene in whole or in part, at an endogenous IL-4Rα locus,with a human IL-4Rα nucleic acid, can be homozygous or heterozygous withrespect to the replacement. In some embodiments, the geneticallymodified rodent is heterozygous with respect to the replacement, i.e.,only one of the two copies of the endogenous rodent IL-4Rα gene has beenreplaced with a human IL-4Rα nucleic acid. In other embodiments, agenetically modified rodent is homozygous with respect to thereplacement, i.e., both copies of the endogenous rodent IL-4Rα gene havebeen replaced with a human IL-4Rα nucleic acid.

The genetically modified rodent disclosed herein expresses a human orhumanized IL-4Rα protein on immune cells, e.g., B and T cells. In someembodiments, the genetically modified rodent does not express endogenousrodent IL-4Rα protein. In one embodiment, the immune cells of the rodentthat expresses a human or humanized IL-4Rα protein have approximatelythe same level of IL-4Rα protein on immune cells as a rodent thatexpresses a functional, endogenous IL-4Rα protein on immune cells of awild-type rodent that expresses a functional, endogenous IL-4Rα proteinand does not express the human or humanized IL-4Rα protein. In otherembodiments, the rodent expresses human or humanized IL-4Rα protein onimmune cells at an amount of at least about 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%,180%, 190% or 200% of the amount of IL-4Rα protein present on immunecells of an age-matched rodent that expresses functional endogenousIL-4Rα protein, but does not comprise a replacement of an endogenousIL-4Rα gene in whole or in part with a human IL-4Rα nucleic acid.

In some embodiments, a genetically modified rodent having a replacementof an endogenous rodent IL-4Rα gene in whole or in part with a humanIL-4Rα nucleic acid and expressing a human or humanized IL-4Rα proteinhas a normal immune system, i.e., the number of immune cells, e.g., Band T cells, in the blood, plasma or serum of the rodent expressinghuman or humanized IL-4Rα protein are similar to the number of immunecells, e.g., B and T cells, in the blood, plasma or serum of a wild-typerodent (e.g., a rodent that expresses functional endogenous IL-4Rαprotein and does not have a replacement of an endogenous rodent IL-4Rαgene in whole or in part with a human IL-4Rα nucleic acid).

In some embodiments, a genetically modified rodent having a replacementof an endogenous rodent IL-4Rα gene in whole or in part with a humanIL-4Rα nucleic acid and expressing a human or humanized IL-4Rα proteinis capable of and functional in mediating IL-4 dependent signaling andIL-13 dependent signaling. For example, a humanized IL-4Rα proteinhaving the ectodomain of a human IL-4Rα protein, expressed on immunecells of a genetically modified rodent, interacts with human IL-4 andmediates human IL-4 dependent signaling via forming Type I receptor (seeFIG. 1). Such humanized IL-4Rα protein having the ectodomain of a humanIL-4Rα protein also interacts with human and mouse IL-13, and mediatesIL-13 dependent signaling via forming Type II receptor (see FIG. 1). Thefunctionality of a humanized IL-4Rα protein expressed in a geneticallymodified rodent can be evaluated in various assays known in the art,including those specifically described in the examples hereinbelow, suchas an assay that measures IL-4 induced IgE class switching using primaryB cells derived from a genetically modified rodent.

In additional embodiments, a genetically modified rodent having areplacement of an endogenous rodent IL-4Rα gene in whole or in part witha human IL-4Rα nucleic acid and expressing a human or humanized IL-4Rαprotein, also includes a replacement of the endogenous rodent IL-4 genein whole or in part, at an endogenous IL-4 locus, with a human IL-4nucleic acid, and as a result, also expresses a human or humanized IL-4.

In a further aspect, doubly humanized rodents, e.g., mice or rats, areprovided whose endogenous rodent IL-4 gene has been replaced in whole orin part, at an endogenous IL-4 locus, with a human IL-4 nucleic acid,and whose endogenous rodent IL-4Rα gene has also been replaced in wholeor in part, at an endogenous IL-4Rα locus, with a human IL-4Rα nucleicacid. Such doubly humanized rodents can be homozygous or heterozygouswith respect to each humanization replacement. In a specific embodiment,the doubly humanized rodent is homozygous with respect to both humanizedIL-4 and humanized IL-4Rα.

The genetic modification to an endogenous rodent IL-4 gene in a doublyhumanized rodent includes those modifications or replacements describedhereinabove for a genetically modified rodent having a replacement of anendogenous rodent IL-4 gene in whole or in part with a human IL-4nucleic acid. Similarly, the genetic modification to an endogenousrodent IL-4Rα gene in a doubly humanized rodent includes thosemodifications or replacements described hereinabove for a geneticallymodified rodent having a replacement of an endogenous rodent IL-4Rα genein whole or in part with a human IL-4Rα nucleic acid. Thus, the featuresdisclosed hereinabove with respect to humanization of the rodent IL-4gene and with respect to humanization of the rodent gene, respectively,are incorporated herein specifically for a doubly humanized rodent.

In specific embodiments, a doubly humanized rodent, e.g., a mouse orrat, is provided that expresses a human IL-4 protein and a humanizedIL-4Rα protein, wherein the humanized IL-4Rα protein includes theectodomain of a human IL-4Rα protein and includes the transmembrane andcytoplasmic domains of the rodent's endogenous IL-4Rα protein. Inparticular embodiments, the expression of the human IL-4 protein and thehumanized IL-4Rα protein are under control of the endogenous rodentregulatory sequences at the endogenous rodent IL-4 locus and rodentIL-4Rα locus, respectively.

In some embodiments, a doubly humanized rodent has a normal immunesystem (i.e., the number of immune cells is approximately the same as awild-type rodent), has approximately the same level of IL-4 protein inthe serum, and expresses approximately the same amount of IL-4Rα proteinon immune cells, as a wild-type rodent, a wild-type rodent being arodent that expresses functional, endogenous IL-4 protein and IL-4Rαprotein and does not express human or humanized IL-4 protein or IL-4Rαprotein.

In particular embodiments, a doubly humanized rodent exhibits afunctional IL-4 signaling pathway. By “functional IL-4 signalingpathway” it is meant that both a human or humanized IL-4 protein, and ahuman or humanized IL-4Rα protein, are expressed in a doubly humanizedrodent and interact with each other in the doubly humanized rodent so asto effectively mediate downstream signal transduction and carry out thebiological activities of a normal IL-4 signaling pathway. The biologicalactivities of a normal IL-4 signaling pathway are described hereinaboveand illustrated in FIG. 1, including those mediated through Type Ireceptor such as initiation and maintenance of the Th2 differentiation,activation and grown of B cells, class switching to IgE and IgG4, andthose mediated through Type II receptor signaling such as Goblet cellhyperplasia, sub-epithelial fibrosis, and tissue remodeling. Forexample, a functional IL-4 signaling pathway in a doubly humanizedrodent is reflected by an inflammatory phenotype characterized by, e.g.,increased IgE in circulation, airway inflammation and/or eosinophilicinfiltrating cells in response to a house dust mite challenge, whichphenotype is also observed in wild-type rodents without the doublehumanization.

Methods of Making a Genetically Modified Non-Human Animal

A genetically modified non-human animal such as a rodent can be madeusing methods known in the art. For example, a targeting vector can bemade that contains a human nucleic acid (such as a human IL-4 or IL-4Rαgene, in whole or in part), flanked by non-human animal homologousupstream and downstream regions. The targeting construct can alsocontain a drug selection cassette (e.g., a floxed hygro selectioncassette, which can be subsequently removed by a transientCre-expression vector), which is positioned 3′ to the human nucleicacid. The targeting vector can be introduced into the genome of anon-human animal cell, e.g., an embryonic stem (ES) cell (such as amouse ES cell) by electroporation, for example. Correctly targeted EScell clones can then be introduced into an early stage embryo (e.g.,8-cell stage mouse embryo). Non-human animals fully derived fromcorrectly targeted ES cells are identified based on, for example, alleleanalysis. For non-human animals where suitable genetically modifiable EScells are not readily available, other methods can be employed to make anon-human animal comprising genetic modifications as described herein.Such methods include, e.g., modifying a non-ES cell genome (e.g., afibroblast or an induced pluripotent cell) and employing nucleartransfer to transfer the modified genome to a suitable cell, e.g., anoocyte, and gestating the modified cell (e.g., the modified oocyte) in anon-human animal under suitable conditions to form an embryo.

Methods of Employing a Genetically Modified Non-Human Animal

In one aspect, genetically modified IL-4 and/or IL-4Rα non-human animalsdisclosed herein are used for evaluating the pharmacodynamics (PD) andtherapeutic efficacy of human-specific IL-4 and/or IL-4Rα antagonists,e.g., neutralizing anti-IL-4 and/or anti-IL-4Rα antibodies (e.g.,dupilumab) in various disease models, as further illustrated in theexamples below.

In some embodiments, the present invention provides a method ofscreening for human-specific IL-4 or IL-4Rα antagonists using a doublyhumanized IL-4 and IL-4Rα mice disclosed herein.

By “IL-4 or IL-4Rα antagonists” it is meant molecules (e.g., antibodies)that block, suppress or inhibit one or more biological functionsmediated by IL-4 or IL-4Rα. “Human-specific IL-4 or IL-4Rα antagonists”refer to antagonists that are specific to the human IL-4 or IL-4Rα, andsubstantially do not act on rodent IL-4 or IL-4Rα.

In specific embodiments, the method of screening utilizes a doublyhumanized mouse that expresses a human IL-4 protein, and a humanizedIL-4Rα protein, wherein the humanized IL-4Rα protein includes theectodomain of a human IL-4Rα protein, linked to the transmembrane andcytoplasmic domains of the endogenous mouse IL-4Rα protein, and whereinthe mouse does not express mouse IL-4 or mouse IL-4Rα.

In some embodiments, the method of screening for a human-specific IL-4or IL-4Rα antagonist comprises administering an agent to a geneticallymodified rodent that is doubly humanized for IL-4 and IL-4Rα asdescribed herein, determining an effect of the agent on a biologicalfunction mediated by the IL-4/IL-4Rα signaling pathway, and identifyingthe agent as a human-specific IL-4 or IL-4Rα antagonist if itantagonizes the function mediated by the IL-4/IL-4Rα signaling pathwayin the genetically modified rodent.

In one embodiment, the agent comprises an immunoglobulin variable domainthat binds IL-4 or IL-4Rα. In one embodiment, the agent specificallybinds human IL-4 or IL-4Rα, but not rodent IL-4 or IL-4Rα. In oneembodiment, the agent is an antibody. In a specific embodiment, theagent is an antibody that specifically binds human IL-4Rα, but notrodent IL-4Rα.

In one embodiment, the method of screening utilizes a doubly humanizedmouse that expresses a human IL-4 protein, and a humanized IL-4Rαprotein, wherein the humanized IL-4Rα protein includes the ectodomain ofa human IL-4Rα protein, linked to the transmembrane and cytoplasmicdomains of the endogenous mouse IL-4Rα protein, and wherein the mousedoes not express murine IL-4 or murine IL-4Rα.

In some embodiments, the method of screening includes the steps ofinducing in a doubly humanized rodent as described herein a diseaseassociated with IL-4/IL-4Rα signaling, administering an agent to therodent, determining whether the agent ameliorates the disease, andidentifying the agent as a human-specific IL-4 or IL-4Rα antagonistsuitable for treating the disease if the agent ameliorates the disease.

By “disease associated with IL-4/IL-4Rα signaling” it is meant a diseasein which the biological function mediated by IL-4/IL-4Rα signaling isimplicated. Examples of diseases associated with IL-4/IL-4Rα signalinginclude, e.g., inflammatory diseases or disorders, such as asthma,atopic dermatitis, chronic obstructive pulmonary disease (COPD) (whichmay result at least in part from cigarette smoke), inflammatory boweldisease, multiple sclerosis, arthritis, allergic rhinitis, eosinophilicesophagitis and psoriasis. Asthma can be eosinophilic ornon-eosinophilic asthma, and steroid sensitive or steroid resistantasthma.

In some embodiments, the disease associated with IL-4/IL-4Rα signalingis airway inflammation, which can be induced in a rodent by intranasaladministration of an allergen (e.g., house dust mite extract) in one ormore doses for a period of time. The effect of an agent can bedetermined by measuring whether the extent of airway inflammation(reflected by e.g., mucus accumulation, eosinophilic infiltrating cellsin bronchoalveolar lavage fluid, levels of total circulating IgE, and/oralteration in expression profile measurable by microarray expressionanalysis) is reduced as a result of the administration of the agent. Theallergen used for inducing airway inflammation and the agent beingtested can be administered simultaneously or at different times. In someembodiments, the allergen is given to the rodent in one or more doses,and the agent being tested is administered to the rodent after at leastone dose of the allergen has been given to the rodent.

In some embodiments, the disease associated with IL-4/IL-4Rα signalingis skin inflammation or atopic dermatitis, which can be induced in arodent by creating skin injury and exposing the injured skin to anallergen (e.g., bacterial toxin or house dust mite extract) in one ormore doses for a period of time. The effect of an agent can bedetermined by measuring whether skin inflammation is reduced as a resultof administration of the agent.

In a further aspect, triply humanized non-human animals, i.e., non-humananimals whose IL-4, IL-4Rα, and IL-33 genes have been humanized, areused to evaluate the pharmacodynamics (PD) and therapeutic efficacy ofcandidate compounds such as, e.g., human-specific IL-4 and/or IL-4Rαantagonists, and human-specific IL-33 antagonists.

By “IL-33 antagonists” it is meant molecules (e.g., antibodies) thatblock, suppress or inhibit one or more biological functions or signalingmediated by IL-33. “Human-specific IL-33 antagonists” refer toantagonists that are specific to the human IL-33, and substantially donot act on rodent IL-33. IL-33 is known to stimulate signal transductionthrough ST2 and IL-1 RAcP, which is diminished in the presence of anantagonist, such as an IL-33 antibody. Inhibition of IL-33 signaltransduction through ST2 and IL-1 RAcP can be determined by assaying forIL-33 signal transduction in an in vitro or in vivo assay, such as thosedescribed in US Published Application 2014/0271658 A1, the entirecontents of which are incorporated herein by reference. For example, anassay such as that described in US Published Application 2014/0271658 A1can be used to assess the effect of an antibody to IL-33 on lunginflammation in allergen-sensitized animals that are homozygous forexpression of human IL-33. An IL-33 antibody that is effective as anIL-33 antagonist should demonstrate a trend towards reduction ininflammatory cells in the lung, as well as a trend towards reduction incytokines such as IL-4 and IL-5.

In specific embodiments, a triply humanized non-human animal is usedherein to evaluate candidate compounds, wherein the triply humanizedanimal is a triply humanized mouse that expresses a human IL-4 protein,a humanized IL-4Rα protein which includes the ectodomain of a humanIL-4Rα protein linked to the transmembrane and cytoplasmic domains of amouse IL-4Rα protein, and a human IL-33 protein, wherein the mouse doesnot express mouse IL-4, mouse IL-4Rα or mouse IL-33.

In some embodiments, a triply humanized non-human animal is used toevaluate the pharmacodynamics (PD) and therapeutic efficacy of acandidate compound, such as, e.g., a human-specific IL-4 and/or IL-4Rαantagonist, or a human-specific IL-33 antagonist. For example, ahuman-specific IL-4 antibody, a human-specific IL-4Rα antibody, and ahuman-specific IL-33 antibody, can be tested individually in a triplyhumanized animal (such as a rodent, e.g., mouse or rat), and their PDprofiles and therapeutic efficacies can be evaluated and compared.

In other embodiments, a triply humanized non-human animal is used toevaluate the efficacy of a combination of compounds, e.g., a combinationof a human specific IL-4 and/or IL-4Rα antagonist antibody, with ahuman-specific IL-33 antagonist antibody, as compared to the efficacy ofthe compounds when used individually to determine, for example, whetherthe combination of compounds exhibits a synergistic therapeutic effect.In specific embodiments, a combination of a human specific IL-4 antibodyand a human-specific IL-33 antibody are tested in a triply humanizednon-human animal. In other specific embodiments, a combination of ahuman specific IL-4Rα antibody and a human-specific IL-33 antibody aretested in a triply humanized non-human animal.

To evaluate a candidate compound or a combination of compounds, adisease associated with the IL-4/IL-4Rα signaling and the IL-33signaling can be induced in the triply humanized animal. Examples ofdiseases associated with the IL-4/IL-4Rα signaling and the IL-33signaling include, e.g., inflammatory diseases or disorders, such asasthma, atopic dermatitis, chronic obstructive pulmonary disease (COPD)(which may result at least in part from cigarette smoke), inflammatorybowel disease, multiple sclerosis, arthritis, allergic rhinitis,eosinophilic esophagitis and psoriasis. Asthma can be eosinophilic ornon-eosinophilic asthma, and steroid sensitive or steroid resistantasthma. The effect of a compound or a combination of compounds can beassessed similarly to an IL-4/IL-4Rα doubly humanized animal asdescribed hereinabove.

The present invention is further illustrated by the following,non-limiting examples.

Example 1

Replacement of the Endogenous Mouse IL-4 Gene with a Human IL-4 Gene

The 8.8 kb human IL-4 gene containing the coding portion of exon 1starting from the ATG initiation codon through exon 4 (including the 3′untranslated region) and a portion of the 3′ region downstream of exon 4of the human IL-4 gene replaced 6.3 kb of the murine IL-4 gene locusspanning the coding portion of exon 1 starting from the ATG initiationcodon through exon 4 (including the 3′ untranslated region) and aportion of the 3′ region downstream of exon 4. See FIG. 2A.

A targeting construct for replacing the mouse with the human IL-4 genein a single targeting step was constructed using VelociGene® geneticengineering technology (see Valenzuela et al. (2003) High-throughputengineering of the mouse genome coupled with high-resolution expressionanalysis, Nature Biotech, 21(6):652-659). Mouse and human IL-4 DNA wereobtained from bacterial artificial chromosome (BAC) clones bMQ-41A12 andRP11-17K19, respectively. Briefly, an Sbfl linearized targetingconstruct generated by gap repair cloning containing mouse IL-4 upstreamand downstream homology arms flanking a 8.8 kb human IL-4 sequenceextending from the ATG codon in exon 1 through exon 4 (including the 3′untranslated region) and a portion of the 3′ region downstream of exon 4(genomic coordinates: GRCh37: chr5:132,009,743-132,018,576 (+strand))and a floxed hygro selection cassette, was electroporated into F1H4mouse embryonic stem (ES) cells (C57BL/6×129 F1 hybrid). Correctlytargeted ES cells (MAID 879) were further electroporated with atransient Cre-expressing vector to remove the drug selection cassette.Targeted ES cell clones without drug cassette (MAID 1553) wereintroduced into an 8-cell stage SW mouse embryo by the VelociMouse®method (see, U.S. Pat. Nos. 7,294,754, 7,576,259, 7,659,442, andPoueymirou et al. (2007) F0 generation mice that are essentially fullyderived from the donor gene-targeted ES cells allowing immediatephenotypic analyses, Nature Biotech. 25(1):91-99). VelociMice® (F0 micefully derived from the donor ES cell) bearing the humanized IL-4 genewere identified by genotyping for loss of mouse allele and gain of humanallele using a modification of allele assay (see, Valenzuela et al.(2003)).

Correctly targeted ES cell clones were identified by aloss-of-native-allele (LONA) assay (Valenzuela et al. 2003) in which thenumber of copies of the native, unmodified IL-4 gene were determined bytwo TaqMan™ quantitative polymerase chain reactions (qPCRs) specific forsequences in the mouse IL-4 gene that were targeted for deletion. TheqPCR assays comprised the following primer-probe sets (written 5′ to3′): upstream forward primer, CATGCACGGA GATGGATGTG (SEQ ID NO:1);upstream reverse primer, GACCCCTCAG GTCCACTTAC C (SEQ ID NO:2); upstreamprobe, FAM-AACGTCCTCA CAGCAACGA-MGB (SEQ ID NO:3); downstream forwardprimer, GTGCCCAGGT GTGCTCATG (SEQ ID NO:4); downstream reverse primer,CGCCTGCCTC CTCACTTTAT C (SEQ ID NO:5); downstream probe, FAM-ATCTGCTTCACCATCCACT-MGB (SEQ ID NO:6); in which FAM refers to the5-carboxyfluorescein fluorescent probe and BHQ refers to thefluorescence quencher of the black hole quencher type (BiosearchTechnologies). DNA purified from ES cell clones that have taken up thetargeting vector and incorporated in their genomes was combined withTaqMan™ Gene Expression Master Mix (Life Technologies) according to themanufacturer's suggestions in a 384-well PCR plate (MicroAmp™ Optical384-Well Reaction Plate, Life Technologies) and cycled in an AppliedBiosystems Prism 7900HT, which collects fluorescence data during thecourse of the PCRs and determines a threshold cycle (Ct), the fractionalPCR cycle at which the accumulated fluorescence reaches a pre-setthreshold. The upstream and downstream IL-4-specific qPCRs and two qPCRsfor non-targeted reference genes were run for each DNA sample. Thedifferences in the Ct values (ΔCt) between each IL-4-specific qPCR andeach reference gene qPCR were calculated, and then the differencebetween each ΔCt and the median ΔCt for all samples assayed wascalculated to obtain ΔΔCt values for each sample. The copy number of theIL-4 gene in each sample was calculated from the following formula: copynumber=2×2⁻ ^(ΔΔ) ^(Ct). A correctly targeted clone, having lost one ofits native copies, will have a IL-4 gene copy number equal to one.Confirmation that the human IL-4 gene sequence replaced the deletedmouse IL-4 gene sequence in the humanized allele was confirmed by aTaqMan™ qPCR assay that comprises the following primer-probe sets(written 5′ to 3′): human forward primer, GCCTGGACCA AGACTCTGT (SEQ IDNO:7); human reverse primer, ACCGTGGGAC GGCTTCTTAC (SEQ ID NO:8); humanupstream probe, FAM-CACCGAGTTG ACCGTAACAG ACATC-BHQ (SEQ ID NO:9).Confirmation that the hygro selection cassette was inserted with thehuman IL-4 gene sequence in the humanized allele was confirmed by aTaqMan™ qPCR assay that comprises the following primer-probe sets(written 5′ to 3′): hygro forward primer, TGCGGCCGAT CTTAGCC (SEQ IDNO:10); hygro reverse primer, TTGACCGATT CCTTGCGG (SEQ ID NO:11); hygroprobe, FAM-ACGAGCGGGT TCGGCCCATT C-BHQ (SEQ ID NO:12).

The same LONA assay was used to assay DNA purified from tail biopsiesfor mice derived from the targeted ES cells to determine their IL-4genotypes and confirm that the humanized IL-4 allele had transmittedthrough the germline. Two pups heterozygous for the replacement are bredto generate a mouse that is homozygous for the replacement of theendogenous mouse IL-4 gene by the human IL-4 gene. Pups that arehomozygous for the replacement are used for phenotyping.

The upstream junction of the murine IL-4 locus and the sequencecontaining the human IL-4 gene is designed to be within 5′-TGCTGATTGGCCCAGAATAA CTGACAATCT GGTGTAATAA AATTTTCCAA TGTAAACTCA TTTTCCCTTGGTTTCAGCAA CTTTAACTCT ATATATAGAG AGACCTCTGC CAGCATTGCA TTGTTAGCATCTCTTGATAA ACTTAATTGT CTCTCGTCAC TGACGGCACA GAGCTATTG(A TGGGTCTCACCTCCCAACTG CTTCCCCCTC TGTTCTTCCT GCTAGCATGT GCCGGCAACT TTGTCCACGGACACAAGTGC GATATCACCT TACAGGAGAT CATCAAAACT TTGAACAGCC TCACAGAGCAGMG)GTGAGT ACCTATCTGG CACCATCTCT CCAGATGTTC TGGTGATGCT CTCAGTATTTCTAGGCATGA AAACGTTAAC AGCTGCTAGA GAAGTTGGAA CTGGTGGTTG GTGGCAGTCCAGGGCACACA GCGAGGCTTC TCCCCTGC (SEQ ID NO:13), wherein the human IL-4sequences are italicized and the IL-4 coding sequences are bracketed.The downstream junction of the sequence containing the human IL-4 geneand the floxed hygro selection cassette is designed to be within5′-TGTTTATTTT GCAG(AATTCC TGTCCTGTGA AGGAAGCCAA CCAGAGTACG TTGGAAAACTTCTTGGAAAG GCTAAAGACG ATCATGAGAG AGAAATATTC AAAGTGTTCG AGCTGA)ATATTTTAATTTAT GAGTTTTTGA TAGCTTTATT TTTTAAGTAT TTATATATTT ATAACTCATCATAAAATAM GTATATATAG AATCTAACAG CAATGGCATT TAATGTATTG GCTATGTTTACTTGACAAAT GAAATTATGG TTTGCAACTT TTAGGGAAAT CAATTTAGTT TACCMGAGACTATAMTGC TATGGGAGCA AAACAGGAAA GACCACTTCC CCCTCGAGGG GTTCCCTCTCGAGTTAGGGA CATAACACAC AAGATAATTA AAGAACACAA GGCCATACAA GATGCGGCCGCACCGGTATA ACTTCGTATA AGGTATCCTA TACGAAGTTA TATGCATGGC CTCCGCGCCGGGTTTTGGCG CCTCCCGCGG GCGCCCCCCT CCTCACGGCG AGCGCTGCCA CGTCAGACGAAGGGCGCAGC GAGCGTCCTG ATCCT (SEQ ID NO:14), wherein the human IL-4sequences are italicized and the IL-4 coding sequences are bracketed.The downstream junction of the sequence of the floxed hygro selectioncassette and the murine IL-4 locus is designed to be within5′-TGCCAAGTTC TAATTCCATC AGACCTCGAC CTGCAGCCGG CGCGCCATAA CTTCGTATAAGGTATCCTAT ACGAAGTTAT CTCGAGAGGA GTTCCCACCC TTCTCAAGAG CATAATGCGCAGATCATTAA GGGACAGATG CAGGCTGGGG AGACGGTTCA GCAGTTAGGA GTACCTGTTGCTCTTCCAGA GGACCCAGGT TCAATTCCCG GCACTCACAT AGCAGCTTAA AACAATAACTCAAGTTCTGG GGGAGCTGAT GCTCTCCTCT GGCCTCCTGT GGAGGTACAC AGACCACATGCCTGTAGGCA AGACACCCAC ACACATAAAA ACAAAATAAA ATAAGGATAG AAAGGCCAGGGGGATGAATC CAGAGGTAGA AGAAAACTTA TTCCCTGGAA TTGTCCTCTG ACTCCCCTCCCAAAACCTCT AACACGCAT (SEQ ID NO:15), wherein the hygro cassettesequences are italicized.

Example 2

Replacement of the Endogenous Mouse IL-4Rα Ectodomain Gene Sequence witha Human IL-4Rα Ectodomain Gene Sequence

The 15.6 kb human IL-4Rα gene containing exon 1 starting from the ATGinitiation codon through exon 5 and a portion of intron 5 of the humanIL-4Rα gene replaced 7.1 kb of the murine IL-4Rα gene locus spanningcoding exon 1 starting from the ATG initiation codon through exon 5 anda portion of intron 5. Mouse exons 6 through 9 were retained; only exons1 through 5 (i.e., the ectodomain) were humanized. See FIG. 2B.

A targeting construct for replacing the mouse with the human IL-4Rα genein a single targeting step was constructed using VelociGene® geneticengineering technology (see Valenzuela et al. (2003) High-throughputengineering of the mouse genome coupled with high-resolution expressionanalysis, Nature Biotech, 21(6):652-659). Mouse and human IL-4Rα DNAwere obtained from bacterial artificial chromosome (BAC) clonesRP23-136G14 and RP11-166E24, respectively. Briefly, a NotI linearizedtargeting construct generated by gap repair cloning containing mouseIL-4Rα gene upstream and downstream homology arms flanking a 15.6 kbhuman IL-4Rα sequence extending from the ATG codon in exon 1 throughexon 5 and a portion of intron 5 (genomic coordinates: GRCh37:chr16:27,351,525-27,367,111 (+ strand)) and a floxed neo selectioncassette, was electroporated into F1H4 mouse embryonic stem (ES) cells(C57BL/6×129 F1 hybrid). Correctly targeted ES cells (MAID 803) werefurther electroporated with a transient Cre-expressing vector to removethe drug selection cassette. Targeted ES cell clones without drugcassette (MAID 1444) were introduced into an 8-cell stage SW mouseembryo by the VelociMouse® method (see, U.S. Pat. Nos. 7,294,754,7,576,259, 7,659,442, and Poueymirou et al. (2007) F0 generation micethat are essentially fully derived from the donor gene-targeted ES cellsallowing immediate phenotypic analyses, Nature Biotech. 25(1):91-99).VelociMice® (F0 mice fully derived from the donor ES cell) bearing thehumanized IL-4Rα gene were identified by genotyping for loss of mouseallele and gain of human allele using a modification of allele assay(see, Valenzuela et al. (2003)).

Correctly targeted ES cell clones were identified by aloss-of-native-allele (LONA) assay (Valenzuela et al. 2003) in which thenumber of copies of the native, unmodified IL-4Rα gene were determinedby two TaqMan™ quantitative polymerase chain reactions (qPCRs) specificfor sequences in the mouse IL-4Rα gene that were targeted for deletion.The qPCR assays comprised the following primer-probe sets (written 5′ to3′): upstream forward primer, CCGCTGGCAT GTGTATTGTG (SEQ ID NO:16);upstream reverse primer, TGAGTGTGGG ACCCTCAAGA G (SEQ ID NO:17);upstream probe, FAM-TGACCCAAGC CCTACATGGC CACT-BHQ (SEQ ID NO:18);downstream forward primer, TGAGGAGAGC TCACGGGAAT C (SEQ ID NO:19);downstream reverse primer, ACCCATCTCC TGCGTTTCTG (SEQ ID NO:20);downstream probe, FAM-TTGACACGCC AGCTACACTG CTCCA-BHQ (SEQ ID NO:21); inwhich FAM refers to the 5-carboxyfluorescein fluorescent probe and BHQrefers to the fluorescence quencher of the black hole quencher type(Biosearch Technologies). DNA purified from ES cell clones that havetaken up the targeting vector and incorporated in their genomes wascombined with TaqMan™ Gene Expression Master Mix (Life Technologies)according to the manufacturer's suggestions in a 384-well PCR plate(MicroAmp™ Optical 384-Well Reaction Plate, Life Technologies) andcycled in an Applied Biosystems Prism 7900HT, which collectsfluorescence data during the course of the PCRs and determines athreshold cycle (Ct), the fractional PCR cycle at which the accumulatedfluorescence reaches a pre-set threshold. The upstream and downstreamIL-4Rα-specific qPCRs and two qPCRs for non-targeted reference geneswere run for each DNA sample. The differences in the Ct values (ΔCt)between each IL-4Rα-specific qPCR and each reference gene qPCR werecalculated, and then the difference between each ΔCt and the median ΔCtfor all samples assayed was calculated to obtain ΔΔCt values for eachsample. The copy number of the IL-4Rα gene in each sample was calculatedfrom the following formula: copy number=2×2⁻ ^(ΔΔ) ^(Ct). A correctlytargeted clone, having lost one of its native copies, will have a IL-4Rαgene copy number equal to one. Confirmation that the human IL-4Rα genesequence replaced the deleted mouse IL-4Rα gene sequence in thehumanized allele was confirmed by a TaqMan™ qPCR assay that comprisesthe following primer-probe sets (written 5′ to 3′): human forwardprimer, ACCTGCGTCT CCGACTACAT G (SEQ ID NO:22); human reverse primer,GAGCTCGGTG CTGCAATTG (SEQ ID NO:23); human probe, FAM-TGGGACCATTCATCTTCCAC TCGCA-BHQ (SEQ ID NO:24). Confirmation that the neo selectioncassette was inserted with the human IL-4Rα gene sequence in thehumanized allele was confirmed by a TaqMan™ qPCR assay that comprisesthe following primer-probe sets (written 5′ to 3′): neo forward primer,GGTGGAGAGG CTATTCGGC (SEQ ID NO:25); neo reverse primer, GAACACGGCGGCATCAG (SEQ ID NO:26); neo probe, FAM-TGGGCACAAC AGACAATCGG CTG-BHQ(SEQ ID NO:27).

The same LONA assay was used to assay DNA purified from tail biopsiesfor mice derived from the targeted ES cells to determine their IL-4Rαgenotypes and confirm that the humanized IL-4Rα allele had transmittedthrough the germline. Two pups heterozygous for the replacement are bredto generate a mouse that is homozygous for the replacement of theendogenous mouse IL-4Rα gene by the human IL-4Rα gene. Pups that arehomozygous for the replacement are used for phenotyping.

The upstream junction of the murine IL-4Rα locus and the sequencecontaining the human IL-4Rα gene is designed to be within 5′-TGGGGGAGGGAGGCCATGAC ACAAATGAAA TGGACCCCGC TGACCCAGGA TCAGCATCTG CCCACTCTTCTTTCTGCAGG CACCTTTTGT GTCCCCA(ATG GGGTGGCTTT GCTCTGGGCT CCTGTTCCCTGTGAGCTGCC TGGTCCTGCT GCAGGTGGCA AGCTCTG)GTA AGTCACCACT TCTCAATCATTCATTTGTTG GCTATTAATG GCGTGCCAGG GTCCTGCAGT ATGTCACCTG GCC (SEQ IDNO:28), wherein the human IL-4Rα sequences are italicized and the IL-4Rαcoding sequences are underlined. The downstream junction of the sequencecontaining the human IL-4Rα gene and the floxed neo selection cassetteis designed to be within 5′-GTCAGATCGT GGAGGGTCTC GGACGAGGG TCCTGACCCTGGGTCTCCAG TCCTGGGAAG TGGAGCCCAG GCTGTACCAT GGCTGACCTC AGCTCATGGCTcccgggctc gataactata acggtcctaa ggtagcgact cgagataact tcgtataatgtatgctatac gaagttatat gcatggcctc cgcgccgggt tttggcgcct cccgcgggcgcccccctcct cacggcgagc gctg (SEQ ID NO:29), wherein the human IL-4Rαsequences are italicized and the cassette sequences are in lower case.The downstream junction of the sequence of the floxed neo selectioncassette and the murine IL-4Rα locus is designed to be within5′-tattgttttg ccaagttcta attccatcag acctcgacct gcagccccta gataacttcgtataatgtat gctatacgaa gttatcctag gttggagctc TCTGTAGCCA GGTAACCAAGGGTCCCAGGG GAACCCCCAG TGTGGACGCG GACTGCACAT GACACAGGGC GGCCTCCCCATTCATGACTG TTTTTCTCCT TGCAG(ACTTC CAGCTGCCCC TGATACAGCG CCTTCCACTGGGGGTCACCA TCTCCTGCCT CTGCATCCCG TTGTTTTGCC TGTTCTGTTA CTTCAGCATTACCAA)GTGAG TTCCTGCTTT GGCTGGTGTC TCTGGCTGGC CCTTCAGCAG TGCTCTCAGAGGTCACAGTC ATTGTGCTGG CTGAGAAAAG (SEQ ID NO:30), wherein the mouseIL-4Rα coding sequences are bracketed, and the neo cassette sequencesare in lower case.

Example 3

Generation of Doubly Humanized IL-4/IL-4Rα Mice

The doubly humanized IL-4/IL-4Rα (Il4^(hu/hu)/Il4ra^(hu/hu)) mice weregenerated as follows. ES cell clone MAID 803, comprising the humanizedIL-4Rα gene and floxed neo cassette, was electroporated with a Creexpression vector to remove the floxed neo cassette to generate ES cellclone MAID 1444, comprising the humanized IL-4Rα gene without a drugselection cassette (see Example 2). The same targeting construct thatwas used to generate ES cell clone MAID 879, comprising the humanizedIL-4 gene and floxed hygro cassette (see Example 1), was electroporatedinto ES cell clone MAID 1444 to generate 879 Het/1444 Het(Il4^(+/hu)/Il4ra^(+/hu)) ES cells, which were subsequentlyelectroporated with a Cre expression vector to remove the floxed hygrocassette to generate an ES cell clone (MAID 1553/1444) comprisinghumanized IL-4 and IL-4Rα genes. The ES cell clone MAID 1553/1444without drug cassette was introduced into an 8-cell stage SW mouseembryo to generate doubly humanized IL-4/IL-4Rα mice.

Example 4

Efficacy Evaluation of Dupilumab, a Fully Human IL-4Rα mAb, in Mice withHuman IL-4 and IL4Rα Gene Replacements

Methods

Genetically engineered mice were created using VelociGene® technology toreplace both mouse full-length IL-4 locus with 8.8 kb of human IL-4genomic sequences (see Example 1 and FIG. 2A) and the extracellulardomain (i.e., ectodomain) of mouse IL-4Rα (CD124) gene with a 15.6 kbfragment of the corresponding human IL-4Rα genomic DNA (see Example 2and FIG. 2B).

Mice with a homozygous humanized IL-4Rα gene were validated forexpression and function of the human gene. To determine the expressionof human IL-4Rα by humanized mice, splenocytes from wild-type andhumanized mice were collected and processed for fluorescent activatedcell sorting (FACS) analysis with fluorescent-labeled antibodies againstmouse CD3, mouse CD19, human CD124, and mouse CD124. (See, e.g., Blaeseret al. (2003) Targeted inactivation of the IL-4 receptor a chain I4Rmotif promotes allergic airway inflammation, J Exp Med198(8):1189-1200).

To demonstrate the ligand specificities and receptor functionalities,primary cells derived from humanized IL-4Rα mice were used. Bonemarrow-derived macrophages were cultured using femoral bone marrow cellsfrom wild-type and humanized IL-4Rα mice in DMEM containing 10% fetalbovine serum plus 20% L-cell conditioned medium for 7 days.

Cells were then treated individually with 20 ng/ml of mouse IL-4, mouseIL-13, human IL-4, human IL-13, or vehicle diluted in culture medium for20 hours. Quadruplicate samples from each condition were harvested forgene expression analysis.

Total RNA from these samples was extracted and amplified into cRNA byincorporating Cy3-CTP. Cy3 labeled cRNA from each sample was thenhybridized to a custom Agilent array comprising of 43,538 60-mer oligoscovering mouse transcriptomes. Data were extracted from scanned arrayimages using Agilent Feature Extraction Software 9.5.

Differentially expressed genes between experimental groups wereidentified using Student's t-test (p<0.05, fold change ≧1.5). Anexpression cluster of these genes was generated using the Pearsoncorrelation clustering algorithm from GeneSpring GX7.3.

The neutralizing effect of dupilumab against IL-4 was evaluated using anin vitro IgE class-switching assay with primary B cells isolated fromhumanized IL-4Rα (Il4ra^(hu/hu)) mice.

Wild-type (WT) and humanized IL-4Rα (Il4ra^(hu/hu)) mice received a highvolume (hydrodynamic) driven gene delivery of naked plasmid DNA solutionfor the expression of mouse IL-25 in vivo. (See, e.g., Liu et al. (1999)Hydrodynamics-based transfection in animals by systemic administrationof plasmid DNA, Gene Therapy 6:1258-1266.) Peripheral blood wascollected 8 days later to measure serum murine IgE (mIgE) levels using acommercial ELISA kit (R & D systems, MN).

Purified primary B cells from humanized mouse splenocytes were activatedwith bacterial LPS and mixed with increasing amounts of recombinanthuman IL-4 in a 7 day culture to induce immunoglobulin class switching.For the antibody blockade experiment, purified B cells were incubatedwith increasing doses of dupilumab for 30 minutes before adding 0.167 nMrecombinant human IL-4 and cultured for 7 days. IgE production in theabsence of IL-4 or with isotype control mAb is shown in (⋄) and (Δ),respectively. The murine IgE levels in the culture supernatants weremeasured using a commercial ELISA kit. (See, e.g., Moon et al. (1989)Regulation of IgG1 and IgE synthesis by interleukin 4 in mouse B cells,Scand I Immunol 30:355-361.)

Interleukin-25 (IL-25) is a cytokine produced by Th2 cells whose mainactivities are mediated through the production of IL-4 and IL-13 toinduce tissue specific pathologies, such as increased pulmonary mucusproduction and goblet cell hyperplasia. (See Fort et al. (2001) IL-25induces IL-4, IL-5, and IL-13 and Th2-associated pathologies in vivo,Immunity 15(6):985-995.)

Lack of IL-13 protects animals from IL-25 induced pathologies in targetorgan. Therefore, an IL-25 driven pulmonary inflammation model was usedto assess the pharmacodynamic (PD) properties of dupilumab in vivo inmice comprising humanized IL-4 and/or IL-4Rα genes.

The PD responses of dupilumab on type II receptors were characterizedusing an IL-25-induced inflammation method by measuring pulmonary mucusaccumulation in the humanized IL-4Rα (Il4ra^(hu/hu)) mice.

On Day 0, WT and humanized IL-4Rα (Il4ra^(hu/hu)) mice received thehydrodynamic delivery of mouse IL-25 expression vector and followed byan injection of dupilumab or isotype control mAb at the indicated doses.Additional doses of antibodies were administered every other day for atotal of 4 doses. On day 8, lung tissues were collected from euthanizedmice and processed lung sections were stained by periodic acid-Schiffbefore blinded scoring for pathological changes.

Results

The humanized IL-4Rα mice were characterized to show: (a) expression ofhuman IL-4Rα on primary cells from doubly humanized IL-4/IL-4Rα(Il4^(hu/hu)/Il4ra^(hu/hu)) mice (see FIG. 3); (b) the change of IL-4ligand specificities in humanized IL-4Rα (Il4ra^(hu/hu)) mice (see FIG.4); and (c) the functionality of the IL-13 pathway in the humanizedIL-4Rα (Il4ra^(hu/hu)) mice (see FIG. 4).

As shown in FIG. 3, in which labeled profiles of IL-4Rα (CD124) on gatedB and T cell populations are shown and the distribution of correspondingunstained cell population is shown in the shaded area, wild-type andhumanized IL-4Rα (Il4ra^(hu/hu)) mice express similar amounts of IL-4Rαprotein on the surface of B (CD19⁺, CD3⁻) and T (CD19⁻, CD3⁺) cells.

As shown in FIG. 4 (left side), wild-type (Il4ra^(+/+)) mice respond tomouse, but not human, IL-4, and respond to both mouse and human IL-13.As shown in FIG. 4 (right side), humanized IL-4Rα (Il4ra^(hu/hu)) micerespond to human, but not mouse, IL-4, and respond to both mouse andhuman IL-13.

This data shows that IL-4, but not IL-13, displays species-specificityin wild-type and humanized IL-4Rα (Il4ra^(hu/hu)) mice.

As shown in FIG. 5, the role of IL-4 as the major factor mediating IgEclass switching was supported by the lack of elevated levels ofcirculating IgE after mouse IL-25 gene delivery in humanized IL-4Rα(Il4ra^(hu/hu)) mice.

The dupilumab monoclonal antibody was investigated in vitro and in vivo.

As shown in FIG. 6, dupilumab prevents human IL-4 induced IgE productionin humanized IL-4Rα (Il4ra^(hu/hu)) mice-derived primary B cellcultures.

As shown in FIG. 7, dupilumab dose dependently reduced IL-25 inducedpulmonary pathologies at 10 mg/kg and above (25 mg/kg reduced mucuspathology

Conclusions

The results demonstrate the pharmacological activity of dupilumab, afully human anti-human IL-4Rα monoclonal antibody, in a geneticallymodified mouse model with cytokine-induced inflammation.

Generation of genetically modified mice with human IL-4 and/or IL-4Rαgene replacements provides a powerful tool to evaluate function of geneorthologs and in vivo efficacy of antibody candidates with limitedcross-species reactivity.

Example 5

House Dust Mite Extract (HDM) Induced Pulmonary Inflammation Model

Chronic airway inflammation in doubly humanized IL-4 and IL-R4α mice isinduced by intranasal challenge of house dust mite (HDM) extract (Greerlaboratories). In brief, mice were first sensitized by intranasalinstillation of HDM suspension (20 μl at the concentration of 2.5 μg/ml)for 10 days. After a two-week interval of resolution, mice werere-challenged with intranasal HDM application 3 times per week betweenweek 5 and 12. The treatment of dupilumab (anti-IL4Rα antibody) wasstarted from the 7th week at the frequency of twice weekly bysubcutaneous injections until the end of experiment at week 12. Tissuesamples were collected for further analyses. The experimental design isdepicted in FIG. 8.

Demonstrating the Therapeutic Efficacy of Dupilumab in the HDM InducedAirway Inflammation Model Using Doubly Humanized IL-4 and IL-4Rα Mice

Airway disease was induced in doubly humanized IL-4 and IL-4Rα(IL-4ra^(hu/hu)/Il4ra^(hu/hu)) mice using the protocol described above.The histological analysis of lung tissue showed that intranasal HDMinstillation caused increased production of mucus in the airway.Treatment of dupilumab reduced the mucus accumulation in the HDMchallenged mice. Analysis of the infiltrating cells in bronchoalveolarlavage fluid (BALF) indicates that the eosinophil counts were increasedby the HDM instillation and were reduced by the treatment of dupilumab.The total circulating IgE was elevated by the treatment of HDM in thehumanized mice, suggesting a competent IL-4 signaling pathway. Use ofdupilumab was capable of reducing the level of IgE. In contrast, acomparator molecule, IL13R2α-Fc, which antagonizes IL-13 only withoutinterfering the IL-4 signal transduction, had comparable activities inreducing mucus accumulation and preventing eosinophil infiltration.Nonetheless, a differential effect was detected in the circulating IgElevel between dupilumab and the IL-13 antagonist, IL13R2α-Fc. Blockadeof IL-13 pathway alone was insufficient to reduce the HDM induced IgElevel; whereas dupilumab reduced the production of IgE, a mainpathogenic mediator of allergy, by blocking both the IL-4 and IL-13pathways.

In a separate set of experiments, airway disease was induced in doublyhumanized IL-4 and IL-4Rα (IL-4^(hu/hu)/IL-4R^(hu/hu)) mice using thesame protocol described above, except that a different control was used.An isotype control antibody of the same IgG isotype as dupilumab wasused in these experiments. The experimental design is depicted in FIG.9. mRNA was purified from total RNA using Dynabeads mRNA kit (Life Tech)and strand specific RNA-Seq library was prepared from mRNA usingScriptseq RNA Library Prep kit (Illumina). The library was sequencedusing HiSeq 2000 (Illumina) at read length of 33 bp and gene expressionlevels were extracted from the raw reads using Clcbio (Qiagen) RNA-Seqworkflow. Differentially expressed genes between experimental groupswere identified using Student's t-test (p<0.05, fold change ≧1.5). Anexpression cluster of these genes was generated using the Pearsoncorrelation clustering algorithm from GeneSpring GX7.3. HDM was found toinduce alteration of pulmonary gene expression in the doubly humanizedIL-4 and IL-4Rα mice and such alteration was blocked by dupilumab. Serumsamples were collected from euthanized mice at the end of the treatmentperiod. The serum murine IgE levels were measured using a commercialELISA kit (R & D systems). Statistical analysis was performed usingordinary one-way ANOVA method.

Example 6

Antigen Induced Cutaneous Inflammation Model

Chronic skin inflammation in doubly humanized Il-4 and Il-4Rα mice canbe induced by the following procedure. The back hair of humanized miceis shaved with electric clipper and then stripped with adhesive tape tocreate minor injuries and break skin barrier. A gauze patch soaked witha solution of allergen (such as ovalbumin plus bacterial toxin or housedust mite extract) is attached to the skin for one week followed by twoweeks of resolution period. The procedure is repeated three times for atotal of 7 weeks to induce atopic dermatitis like skin lesions. Thetreated mice will have increased IgE levels, pruritis, thickening of theepidermis, typical symptoms of atopic dermatitis.

Example 7

Characterizing PK Profiles of Anti-Human IL-4Rα Antibodies in MiceExpressing Humanized IL-4Rα

This Example describes experiments conducted to evaluate the PK profilesof REGN 668 (human monoclonal antibody directed to human IL-4Rα, alsoknown as “dupilumab”) and control antibody REGN646 (monkey surrogate,anti-mfIL-4R non-binding control antibody).

The mice used in these experiments were MAID 1444 (homozygous forhumanized IL-4Rα, or “IL-4Rα HumIn”, in which the IL-4Rα ectodomain ishuman and the transmembrane and cytoplasmic regions are mouse) andstrain-matched (75% C57BL/6/25%129Sv) wild-type (“WT”) mice of 20-23weeks. The study group included a total of 40 mice, male and female,with a cohort size per drug/per dose of 5 homozygous and 5strain-matched WT. The antibodies (in PBS buffer) were given to mice viasubcutaneous injection at 10 mg/kg. Blood samples were taken foranalysis on the day of the injection (time point “0” or day 0), at 6 hrpost injection, and on day 1, day 3, day 7, day 10, day 14, day 21, andday 30, respectively.

The circulating drug (i.e., REGN668 or REGN646) levels were determinedby total human antibody analysis using an ELISA immunoassay. Briefly, agoat anti-human IgG polyclonal antibody (Jackson ImmunoResearch,#109-005-098) was coated onto 96-well plates to capture the tested humanantibodies in the sera, and then plate-bound antibodies were detectedusing a goat anti-human IgG polyclonal antibody conjugated withhorseradish peroxidase (Jackson ImmunoResearch, #109-035-098) and TMBsubstrate (BD Pharmingen). The serum samples were in six-dose serialdilutions per sample ranging from 1:100-1:243,000 and referencestandards of the respective antibodies were in 12-dose serial dilutions.Drug antibody concentrations in the sera were calculated based on thereference standard curve generated using Graphpad Prism software.

The half-life of REGN 668 was found to be shortened in IL-4Rα HumIn miceas compared to wild-type mice with only mouse IL-4Rα protein. Thisdifference in PK profiles could be explained by the target mediatedinteraction and clearance between monoclonal antibodies and human IL-4αreceptor. Therefore, mice expressing human or humanized IL-4Rα providesuitable simulation to characterize the PK properties of anti-humanIL-4Rα antibodies (e.g., dupilumab) in a preclinical mouse model.

Uses for Humanized IL-4 and/or IL-4Rα Mice

Humanized IL-4 and/or IL-4Rα are useful to evaluate the pharmacodynamics(PD) of human-specific IL-4 and/or IL-4Rα antagonists, e.g.,neutralizing anti-IL-4 and/or or anti-IL-4Rα antibodies, e.g.,dupilumab.

Pharmacokinetics (PK) and PD assays in humanized IL-4 and/or IL-4Rα miceare performed according to standard procedures known in the art.

Humanized IL-4 and/or IL-4Rα mice are useful to test the in vivotherapeutic efficacy of human-specific IL-4 and/or IL-4Rα antagonists,e.g., neutralizing anti-IL-4 and/or IL-4Rα antibodies, e.g., dupilumab,in a variety of disease models known in the art, e.g., as shownhereinabove.

Example 8

Replacement of the Endogenous Mouse IL-33 Gene with a Human IL-33 Gene

The mouse IL-33 gene (NCBI Gene ID: 77125, Primary source: MGI:1924375;RefSeq transcript: NM_001164724.1; UniProt ID: Q8BVZ5; Genomic assembly:NCBI37/mm9; Location: chr19:29,999,604-30,035,205+ strand) has 8 exonsand encodes a protein of 266 amino acids (GenBank Accession No.NP_001158196.1).

The human IL-33 gene (NCBI Gene ID: 90865, Primary source: HGNC:16028;RefSeq transcript: NM_033439.3; UniProt ID: 095760; Genomic assembly:GRCh37/hg19; Location: chr9:6,215,149-6,257,983+ strand) also has 8exons and encodes a protein of 270 amino acids (GenBank Accession No.NP_254274.1).

A 16333 bp human genomic segment containing exon 2 starting from the ATGinitiation codon through exon 8 (including the 3′ untranslated region)of the human IL-33 gene replaced 9381 bp of the mouse IL-33 gene locusspanning exon 2 starting from the ATG initiation codon through thecoding portion of exon 8 including the stop codon. See FIG. 10A.

A targeting construct for replacing the mouse IL-33 gene with a humanIL-33 genomic segment in a single targeting step was constructed usingVelociGene® genetic engineering technology (see Valenzuela et al. (2003)High-throughput engineering of the mouse genome coupled withhigh-resolution expression analysis, Nature Biotech, 21(6):652-659),similar to the procedure described in Example 1 above for replacing themouse IL-4 gene with a human IL-4 genomic segment, except that mouse andhuman IL-33 DNA were obtained from bacterial artificial chromosome (BAC)clones bMQ-350I18 and CTD-3015M15, respectively, and that the targetingvector contained a loxP neomycin selection cassette (FIG. 10B).

Correctly targeted ES cell clones (MAID 7060) were identified by aloss-of-native-allele (LONA) assay (Valenzuela et al. 2003) in which thenumber of copies of the native, unmodified IL-33 gene were determined bytwo TaqMan™ quantitative polymerase chain reactions (qPCRs) specific forsequences in the mouse IL-33 gene that were targeted for deletion. TheqPCR assays comprised the following primer-probe sets (written 5′ to3′):

upstream (“mTU”): (SEQ ID NO: 31)forward primer, TTGGACTAGTAACAAGAAGGGTAGCA; (SEQ ID NO: 32)reverse primer, CCTTTCCCATCACCCTCTAACTT; (SEQ ID NO: 33)probe (MGB), AGCTCTGGTGGACAGA; downstream (“mTD”): (SEQ ID NO: 34)forward primer, TCTCTGCCAAGCTGCTTATCC; (SEQ ID NO: 35)reverse primer, GGCTGCATGGAAGAGGTGAA; (SEQ ID NO: 36)probe (MGB), CTCTCCACAAATCG.

Confirmation that the human IL-33 gene sequence replaced the mouse IL-33gene sequence in the humanized allele was confirmed by a TaqMan™ qPCRassay that comprises the following primer-probe sets (written 5′ to 3′):

upstream (“hTU”) (SEQ ID NO: 37)forward primer, CAGGCAGGAATAGCTGAGATAATCT;  (SEQ ID NO: 38)reverse primer, TGTGGAGCAAAAAGTGGTTGAT;  (SEQ ID NO: 39)probe (MGB), CCTGTGAATAGTGATAAAC;  downstream (“hTD”): (SEQ ID NO: 40)forward primer, CAGTTCCAAACGATAGGCTCAA;  (SEQ ID NO: 41)reverse primer, ATAATTCTGTGAAGCATCTGGTCTTC;  (SEQ ID NO: 42)probe (MGB), CTAGAGCTGCTAGTAAAA. 

The upstream junction of the murine IL-33 locus and the sequencecontaining the human IL-33 gene (shown as “I” in FIG. 10B) is designedto be within 5′-ATAGCCAAGG TTGCTTCTGA TGACTTCAGG TCCATATAGT TGGATTAATGTTATATTTCA ATCCCACAGA AACCTGAAAA

AAGCCTA AAATGAAGTA TTCAACCAAC AAAATTTCCA CAGCAAAGTG GAAGAACACAGCAAGCAAAG CCTTGTGTTT-3′ (SEQ ID NO: 43), wherein the human IL-33sequence is italicized and the human start codon ATG is underlined. Thedownstream junction of the sequence containing the human IL-33 genomicsequence and the loxP neomycin selection cassette (shown as “II” in FIG.10B) is designed to be within

(SEQ ID NO: 44) 5'-TTTATATTAT TGAATAAAGT ATATTTTCCA AATGTATGTG AGACTATAAT GATTTTATCA TATGATGACT CAATATTCTG/CTCGAGATAA CTTCGTATAA TGTATGCTAT ACGAAGTTATATGCATGGCC TCCGCGCCGG GTTTTGGCGC CTCCCGCGGG-3',wherein the human IL-33 sequence is italicized and the junction isindicated by the “/” symbol, and the lox P site is underlined. Thedownstream junction of the sequence of the loxP neo selection cassetteand the murine IL-33 locus (shown as “III” in FIG. 10C) is designed tobe within

(SEQ ID NO: 45) 5'-AGCCCCTAGA TAACTTCGTA TAATGTATGC TATACGAAGTTATGCTAGTA ACTATAACGG TCCTAAGGTA GCGAGCTAGC/CGCCTGTGCG TTCTGGGTTG AATGACTTAA TGCTTCCAACTGAAGAAAGG GTAACAGAGA GAAAGAAAGC CATTCTTGGC-3', wherein the junction is shown by the “/” symbol, and the loxP site isunderlined.

Correctly targeted ES cells (MAID 7060) were further electroporated witha transient Cre-expressing vector to remove the drug selection cassetteand obtain ES cell clones without drug cassette (MAID 7061). Theupstream junction in these MAID 7061 ES cells (shown as “I” in FIG. 18C)is the same as in MAID 7060 ES cells. The downstream junction (shown as“II” in FIG. 18C) is designed to be within

(SEQ ID NO: 46), 5'-TTTATATTAT TGAATAAAGT ATATTTTCCA AATGTATGTG AGACTATAAT GATTTTATCA TATGATGACT CAATATTCTG/CTCGAGATAA CTTCGTATAA TGTATGCTAT ACGAAGTTAT GCTAGTAACT ATAACGGTCC TAAGGTAGCG AGCTAGC/CGCCTGTGCG TTCTGGGTTG AATGACTTAA TGCTTCCAAC  TGAAGAAAGG GTAACAGAGA GAAAGAAAGC CATTCTTGGC-3'wherein the 3′ human IL-33 sequence is italicized before the first “/”symbol, and the mouse IL-33 3′ sequence is italicized after the second“/” symbol, and the loxP site is underlined.

Correctly targeted ES cells (MAID 7060 or MAID 7061) were introducedinto an 8-cell stage SW mouse embryo by the VelociMouse® method (see,U.S. Pat. Nos. 7,294,754, 7,576,259, 7,659,442, and Poueymirou et al.(2007) F0 generation mice that are essentially fully derived from thedonor gene-targeted ES cells allowing immediate phenotypic analyses,Nature Biotech. 25(1):91-99). VelociMice® (F0 mice fully derived fromthe donor ES cell) bearing the humanized IL-33 gene were identified bygenotyping for loss of mouse allele and gain of human allele using amodification of allele assay (see, Valenzuela et al. (2003)). The sameLONA assay was used to assay DNA purified from tail biopsies for micederived from the targeted ES cells to determine their IL-33 genotypesand confirm that the humanized IL-33 allele had transmitted through thegermline. Two pups heterozygous for the replacement were bred togenerate a mouse that is homozygous for the replacement of theendogenous mouse IL-33 gene by the human IL-33 gene.

What is claimed is:
 1. A rodent, comprising a replacement of a genomicDNA of a rodent IL-4Rα gene at an endogenous rodent IL-4Rα locus with ahuman genomic DNA of a human IL-4Rα gene to form a humanized IL-4Rαgene, wherein the genomic DNA of the rodent IL-4Rα gene comprises theATG initiation codon of exon 1 through exon 5 of the rodent IL-4Rα gene,and the human genomic DNA of the human IL-4Rα gene comprises the ATGinitiation codon of exon 1 through exon 5 of the human IL-4Rα gene;wherein the humanized IL-4Rα gene comprises the ATG initiation codon ofexon 1 through exon 5 of the human IL-4Rα gene and exons 6-9 of therodent IL-4Rα gene, and the human genomic DNA present in the humanizedIL-4Rα gene begins with the ATG initiation codon in exon 1 of the humanIL-4Rα gene; and wherein expression of the humanized IL-4Rα gene isunder control of the rodent IL-4Rα promoter at the endogenous rodentIL-4Rα locus, wherein the rodent is a mouse or a rat.
 2. The rodent ofclaim 1, wherein the rodent is a mouse that is incapable of expressing amouse IL-4Rα protein.
 3. The rodent of claim 1, wherein the rodentexpresses a human or humanized IL-4 protein.
 4. The rodent of claim 3,wherein the rodent comprises a replacement of a genomic DNA of a rodentIL-4 gene at an endogenous rodent IL-4 locus with a genomic DNA of ahuman IL-4 gene to form a humanized IL-4 gene, wherein the genomic DNAof the rodent IL-4 gene comprises the ATG initiation codon of exon 1through exon 4 of the rodent IL-4 gene, and the genomic DNA of the humanIL-4 gene comprises the ATG initiation codon of exon 1 through exon 4 ofthe human IL-4 gene, and wherein expression of the humanized IL-4 geneis under control of the rodent IL-4 promoter at the endogenous rodentIL-4 locus.
 5. The rodent of claim 1, wherein the rodent is a mouse. 6.A method for making a humanized rodent, comprising replacing a genomicDNA of a rodent IL-4Rα gene at an endogenous rodent IL-4Rα locus with ahuman genomic DNA of a human IL-4Rα gene to form a humanized IL-4Rαgene, wherein the genomic DNA of the rodent IL-4Rα gene comprises theATG initiation codon of exon 1 through exon 5 of the rodent IL-4Rα gene,and the human genomic DNA of the human IL-4Rα gene comprises the ATGinitiation codon of exon 1 through exon 5 of the human IL-4Rα gene;wherein the humanized IL-4Rα gene comprises the ATG initiation codon ofexon 1 through exon 5 of the human IL-4Rα gene followed by exons 6-9 ofthe rodent IL-4Rα gene, and the human genomic DNA present in thehumanized IL-4Rα gene begins with the ATG initiation codon of exon 1 ofthe human IL-4Rα gene; and wherein the humanized IL-4Rα gene is operablylinked to the rodent IL-4Rα promoter at the endogenous rodent IL-4Rαlocus, wherein the rodent is a mouse or a rat.
 7. The method of claim 6,wherein the rodent is a mouse.
 8. The rodent of claim 3, wherein therodent is a mouse that does not express a mouse IL-4 protein.